Method of preparing organs for vitrification

ABSTRACT

The invention relates to the field of organ and tissue perfusion. More particularly, the present invention relates to a method for preparing organs, such as the kidney and liver, for cryopreservation through the introduction of vitrifiable concentrations of cryoprotectant into them. To prepare the organ for cryopreservation, the donor human or animal, is treated in the usual manner and may also be treated with iloprost, or other vasodilators, and/or transforming growth factor β1. Alternatively, or additionally, the organ which is to be cryopreserved can be administered iloprost, or other vasodilators, and/or transforming growth factor β1 directly into its artery. The invention also relates to preparing organs for transplantation by a method for the removal of the cryoprotectant therefrom using low (such as raffinose, sucrose, mannitol, etc.), medium (such as agents with intermediate molecular weights of around 600-2,000) and high (such as hydroxyethyl starch) molecular weight agents osmotic buffering agents. The invention is also directed to new post-transplantation treatments such as the use of transforming growth factor β1, N-acetylcysteine and aurothioglucose. Further, the invention relates to a method of vitrification of organs comprising a multi-step method of introducing increasing concentrations of cryoprotectant with osmotic equilibration of the organ and decreases in temperature prior to perfusion with vitrifiable concentrations of cryoprotectant at about -5° C. to -35° C.

RIGHTS OF THE UNITED STATES GOVERNMENT IN THIS INVENTION

This invention was made with United States Government support underNational Institutes of Health Grant Nos. GM 1759, BSRG 2507 and RR05737. The United States Government has certain rights in thisinvention.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a Continuation in Part (CIP) of U.S. patentapplication Ser. No. 08/072,754, filed Jun. 7, 1993, now abandoned,which is a CIP of application Ser. No. 07/725,054, filed on Jul. 8, 1991(issued on Jun. 8, 1993 as U.S. Pat. No. 5,217,860). This application isalso related to U.S. patent application Ser. No. 08/029,432, nowabandoned, which was filed on Mar. 10, 1993 and is a divisional of Ser.No. 07/725,054 now U.S. Pat. No. 5,217,860. This application is alsorelated to U.S. Pat. No. 4,559,298.

FIELD OF THE INVENTION

This invention relates to the field of organ perfusion. Moreparticularly, it relates to a computer controlled apparatus and methodfor perfusing isolated animal, including human, organs. Still moreparticularly, this invention relates to an apparatus and methods forintroducing vitrifiable concentrations of cryoprotective agents intoisolated organs or tissues in preparation for their cryopreservation andfor removing these agents from the organs and tissues after theircryopreservation in preparation for their transplantation into ananimal, including into a human.

BACKGROUND OF THE INVENTION

Cryopreservation (that is, preservation at very low temperatures) oforgans would allow organ banks to be established for use by transplantsurgeons in much the same way that blood banks are used by the medicalcommunity today. At the present time, cryopreservation can be approachedby freezing an organ or by vitrifying the organ. If an organ is frozen,ice crystals form within the organ which mechanically disrupt itsstructure and hence damage its ability to function correctly when it istransplanted into a recipient. Vitrification, by contrast, meanssolidification, as in a glass, without ice crystal formation.

The main difficulty with cryopreservation is that it requires theperfusion of organs with high concentrations of cryoprotective agents(water soluble organic molecules that minimize or prevent freezinginjury during cooling to very low temperatures). No fully suitableequipment or method(s) has been developed to date for carrying out thisperfusion process. This has prevented the establishment of viable organbanks that could potentially save lives.

Devices and methods for perfusing organs with cryoprotectant have beendescribed in the literature since the early 1970's. See, Pegg, D. E., inCurrent Trends in Cryobiology (A. U. Smith, editor) Plenum Press, NewYork, N.Y., 1970, pp. 153-180, but particularly pages 175-177; and Pegg,D. E., Cryobiology 9: 411-419 (1972).

In the apparatus initially described by Pegg, two perfusion circuitsoperated simultaneously, one with and one without cryoprotectant.Cryoprotectant was introduced and removed by abruptly switching from thecryoprotectant-free circuit to the cryoprotectant-containing circuit,then back again. The pressure was controlled by undescribed techniques,and data was fed into a data logger which provided a paper tape outputwhich was processed by a programmable desk-top Wang calculator. Theexperimental results were poor. The equipment and technique describedwere considered inadequate by Pegg and his colleagues, who latermodified them considerably.

In 1973, Sherwood et al. (in Organ Preservation, D. E. Pegg, ed.,Churchill Livingstone, London (1973), pp. 152-174), described fourpotential perfusion systems, none of which are known to have been built.The first system consisted of a family of reservoirs connected directlyto the organ via a multiway valve, changes being made in steps simply byswitching from one reservoir to another.

The second system created changes in concentration by metering flow froma diluent reservoir and from a cryoprotectant concentrate reservoir intoa mixing chamber and then to the kidney. No separate pump forcontrolling flow to the kidney was included. Total flow was controlledby the output of the metering pumps used for mixing. A heat exchangerwas used before rather than after the filter (thus limiting heatexchanger effectiveness), and there was an absence of most arterialsensing. As will become readily apparent below, the only similaritybetween this system and the present invention was the use of twoconcentration sensors, one in the arterial line and one in the venousline of the kidney. Organ flow rate was forced to vary in order tominimize arteriovenous (A-V) concentration differences. The sensing ofconcentration before and after the kidney in the circuit is analogous tobut substantially inferior to the use of a refractometer and adifferential refractometer in the present invention. The presentinventors' experience has shown that the use of a differentialrefractometer is necessary for its greater sensitivity. The concept ofcontrolling organ A-V gradient by controlling organ flow is distinctlyinferior to the system of the present invention.

The third system described by Sherwood et al. also lacked a kidneyperfusion pump, relying on a "backpressure control valve" to recirculateperfusate from the filter in such a way as to maintain the desiredperfusion pressure to the kidney. As with the second Sherwood system,the heat exchanger is proximal to the filter and no bubble trap ispresent. The perfusate reservoir's concentration is controlled bymetered addition of cryoprotectant or diluent as in the second Sherwoodsystem, and if flow from the organ is not recirculated, major problemsarise in maintaining and controlling perfusate volume and concentration.None of these features is desirable.

The fourth system was noted by Pegg in an appendix to the main paper. Inthis system, perfusate is drained by gravity directly from the mixingreservoir to the kidney through a heat exchanger, re-entering thereservoir after passing through the kidney. Concentration is sensed alsoby directly and separately pumping liquid from the reservoir to therefractometer and back.

Modifications and additional details were reported by Pegg et al.(Cryobiology 14: 168-178 (1977)). The apparatus used one mixingreservoir and one reservoir for adding glycerol concentrate orglycerol-free perfusate to the mixing reservoir to controlconcentration. The volume of the mixing reservoir was held constantduring perfusion, necessitating an exponentially increasing rate ofdiluent addition during cryoprotectant washout to maintain a linear rateof concentration change. The constant mixing reservoir volume and thepresence of only a single delivery reservoir also made it impossible toabruptly change perfusate concentration. All components of the circuitother than the kidney and a pre-kidney heat exchanger were located on alab bench at ambient temperature, with the reservoir being thermostatedat a constant 30° C. The kidney and the heat exchanger were located in astyrofoam box whose internal temperature was not controlled. Despitethis lack of control of the air temperature surrounding the kidney, onlythe arterial temperature but not the venous temperature or even thekidney surface temperature was measured. The use of a styrofoam box alsodid not allow for perfusion under sterile conditions. The only possibleway of measuring organ flow rate was by switching off the effluentrecirculation pump and manually recording the time required for a givenvolume of fluid to accumulate in the effluent reservoir, since there wasno perfusion pump which specifically supplied the organ, unlike thepresent invention. Pressure was controlled, not on the basis of kidneyresistance, but on the basis of the combined resistance of the kidneyand a manually adjustable bypass valve used to allow rapid circulationof perfusate through the heat exchanger and back to the mixingreservoir. The pressure sensor was located at the arterial cannula,creating a fluid dead space requiring manual cleaning and potentiallyintroducing undesired addition of unmixed dead space fluid into thearterial cannula. Pressure control was achieved by means of aspecially-fabricated pressure control unit whose electrical circuit wasdescribed in an earlier paper (Pegg et al., Cryobiology 10: 56-66(1973)). Arterial concentration but not venous concentration wasmeasured. No computer control or monitoring was used. Concentration wascontrolled by feeding the output of the recording refractometer into a"process controller" for comparison to the output of a linear voltageramp generator and appropriate adjustment of concentrate or diluent flowrate. Glycerol concentrations were measured manually at 5 minuteintervals at both the mixing reservoir and the arterial sample port:evidently, the refractometer was not used to send a measurable signal toa recording device. Temperature and flow were recorded manually at 5minute intervals. Arterial pressure and kidney weight were recorded aspen traces on a strip chart recorder. None of these features isdesirable.

Further refinements were reported by Jacobsen et al. (Cryobiology 15:18-26 (1978)). A bubble trap was added, the sample port on the kidneybypass was eliminated (concentration was measured at the distal end ofthe bypass line instead), and temperature was recorded as a trace on astrip chart recorder rather than manually every 5 minutes. Additionally,these authors reported that bypass concentration lagged reservoirconcentration by 5 min (v. 3 min or less for arterial concentration inthe present invention) and that terminal cryoprotectant concentrationcould not be brought to less than 70 mM after adding 5 liters of diluentto the mixing reservoir (v. near-zero terminal concentrations in thepresent invention using less than 3 liters of diluent and using peakcryoprotectant concentrations approximately twice those of Jacobsen etal., supra).

A variation on the system was also reported the same year by I. A.Jacobsen (Cryobiology 15: 302-311 (1978)). Jacobsen measured but did notreport air temperatures surrounding the kidney during perfusion. Hereduced the mixing reservoir volume to 70 ml, which was a small fractionof the 400 ml total volume of the circuit. No electronic-outputrefractometer appears to have been used to directly sense glycerolconcentration and control addition and washout. Instead, the calculatedvalues of concentrate or diluent flow rate were drawn on paper withIndia ink and read by a Leeds and Northrup Trendtrak Programmer whichthen controlled the concentrate/diluent pump. Despite the low circuitvolume, the minimum concentration of cryoprotectant which could beachieved was about 100 mM.

Additional alterations of the same system were reported by Armitage etal. (Cryobiology 18: 370-377 (1981)). Essentially, the entire perfusioncircuit previously used was placed into a refrigerated cabinet. Insteadof a voltage ramp controller, a cam-follower was used. Again, however,it was necessary to calculate the required rates of addition of glycerolor diluent using theoretical equations in order to cut the cam properly,an approach which may introduce errors in the actual achievement of thedesired concentration-time histories. Finally, a modification was madein which an additional reservoir was added to the circuit. Thisreservoir was apparently accessed by manual stopcocks (the mode ofswitching to and from this reservoir was not clearly explained), and useof the new reservoir was at the expense of being able to filter theperfusate or send it through a bubble trap. The new reservoir was notused to change cryoprotectant concentration; rather, it was used tochange the ionic composition of the medium after the cryoprotectant hadbeen added. The volume of the mixing reservoir was set at 500 ml,allowing a final cryoprotectant concentration of 40 mM to be achieved.

To the best of the inventors' knowledge, the devices and methodsdescribed above represent the current state of the art of cryoprotectantperfusion as practiced by others.

An approach to organ preservation at cryogenic temperatures previouslydescribed by one of the Applicants involved vitrifying rather thanfreezing organs during cooling (see, for example, Fahy et al.,Cryobiology 21: 407-426 (1984); and U.S. Pat. No. 4,559,298)."Vitrification" means solidification without freezing and is a form ofcryopreservation. Vitrification can be brought about in living systems,such as isolated human or other animal organs, by replacing largefractions of the water in these systems with cryoprotective agents (alsoknown as cryoprotectants) whose presence inhibits crystallization ofwater (i.e., ice formation) when the system or organ is cooled.Vitrification typically requires concentrations greater than 6 molar (M)cryoprotectant. However, using known techniques, it has not beenpossible to use sufficiently high cryoprotectant concentrations tovitrify an organ without killing it. The limiting concentration fororgan survival was typically just over 4M.

One type of damage caused by cryoprotectants is osmotic damage.Cryobiologists learned of the osmotic effects of cryoprotectants in the1950's and of the necessity of controlling these effects so as toprevent unnecessary damage during the addition and removal ofcryoprotectants to isolated cells and tissues. Similar lessons werelearned when cryobiologists moved on to studies of whole organ perfusionwith cryoprotectants. Attention to the principles of osmosis wereessential to induce tolerance to cryoprotectant addition to organs.Despite efforts to control the deleterious osmotic effects ofcryoprotectants, limits of tolerance to cryoprotectants are stillobserved. There appear to be genuine, inherent toxic effects ofcryoprotectants that are independent of the transient osmotic effects ofthese chemical agents.

Studies by the present inventors and others have examined methods ofcontrolling the non-osmotic, inherent toxicity of cryoprotective agents.The results indicate that several techniques can be effective alone andin combination. These include (a) exposure to the highest concentrationsat reduced temperatures; (b) the use of specific combinations ofcryoprotectants whose effects cancel out each other's toxicities; (c)exposure to cryoprotectants in vehicle solutions that are optimized forthose particular cryoprotectants; (d) the use of non-penetrating agentsthat can substitute for a portion of the penetrating agent otherwiseneeded, thus sparing the cellular interior from exposure to additionalintracellular agent; and (e) minimization of the time spent within theconcentration range of rapid time-dependent toxicity. Means by whichthese principles could be applied to whole organs so as to permit themto be treated with vitrifiable solutions without perishing, however,have not been clear or available.

Some of these techniques are in potential conflict with the need tocontrol osmotic forces. For example, reduced temperatures also reducethe influx and efflux rate of cryoprotectants, thereby prolonging andintensifying their osmotic effects. Similarly, minimizing exposure timeto cryoprotectants maximizes their potential osmotic effects. Thus,there must be a balance reached between the control of osmotic damageand the control of toxicity. Adequate means for obtaining this balancehave not been described in the literature. In some cases, intensifyingthe osmotic effects of cryoprotectants by minimizing exposure times tothese agents can be beneficial and complementary to the reduced toxicitythat results, but safe means for achieving this in whole organs have notbeen described.

Organ preservation at cryogenic temperatures would permit the reductionof the wastage of valuable human organs and would facilitate bettermatching of donor and recipient, a factor which continues to beimportant despite the many recent advances in controlling rejection(see, Takiff et al., Transplantation 47: 102-105 (1989); Gilks et al.,Transplantation 43: 669-674 (1987)). Furthermore, most techniques nowbeing explored for inducing recipient immunological tolerance of aspecific donor organ would be facilitated by the availability of moretime for recipient preparation.

One major limitation in organ cryopreservation studies has been the lackof suitable equipment for controlling perfusion parameters such ascryoprotectant concentration-time history, pressure, and temperature.Previously described standard perfusion machines are not designed forthis application and are unable to meet the requirements addressed here.Patented techniques heretofore known are described in:

U.S. Pat. No. 3,753,865 to Belzer et al.;

U.S. Pat. No. 3,772,153 to De Roissart et al.;

U.S. Pat. No. 3,843,455 to Bier, M.

U.S. Pat. No. 3,892,628 to Thorne et al.;

U.S. Pat. No. 3,914,954 to Doerig, R. K.;

U.S. Pat. No. 3,995,444 to Clark et al.;

U.S. Pat. No. 4,629,686 to Gruenberg, M. L.; and

U.S. Pat. No. 4,837,390 to Reneau, R. P.

Equipment described for cryopreservation applications in the past haspermitted only relatively simple experimental protocols to be carriedout, and has often been awkward to use. Only Adem et al. have reportedusing a computer for organ perfusion with cryoprotectant (see, forexample, J. Biomed. Engineering 3: 134-139 (1981)). However, theirspecific design has several major flaws that limit its utility.

The present invention overcomes substantially all of the deficiencies ofknown apparatus and methods.

SUMMARY OF THE INVENTION

In one embodiment, the present invention is directed to acomputer-controlled apparatus and methods for perfusing a human or otheranimal organ, such as a kidney, liver, heart, etc., with a perfusate,and may include preparing the organ for such perfusion. The perfusion ofthe organ may be done for any one of a number of reasons including, butnot limited to, for example: to prepare the organ for cryopreservation;to prepare the organ for transplantation after its cryopreservation; topreserve it by conventional means above 0° C.; to keep it alivetemporarily at high temperatures to study its physiology; to test theorgan's viability; to attempt resuscitation of the organ; and to fix theorgan for structural studies. The apparatus and methods may also be usedto superfuse an organ or tissue slice. In another embodiment, thisinvention is directed to the treatment of the donor animal and/or theabout-to-be donated organ with iloprost and/or other drugs to prepare itfor perfusion. In another embodiment, this invention is directed to anapparatus and method which is used to prepare the organ forcryopreservation, such as by vitrification. In another embodiment, thisinvention is directed to an apparatus and methods for preparing an organfor transplantation into an appropriate host after its cryopreservation.

In one embodiment, this invention is directed to a method of preparing abiological organ for cryopreservation, comprising the steps of:

(a) perfusing the organ with gradually increasing concentrations ofcryoprotectant solution to a first predetermined concentration whileconcurrently reducing the temperature of the organ;

(b) maintaining the concentration of the cryoprotectant for a sufficienttime to permit the approximate osmotic equilibration of the organ tooccur; and

(c) increasing the cryoprotectant concentration of the solution to ahigher second predetermined concentration and maintaining thecryoprotectant concentration of the solution at the second concentrationfor a time sufficient to permit the approximate osmotic equilibration ofthe organ to occur.

The organ is then removed from the perfusion apparatus and iscryopreserved using an appropriate method or is further prepared forcryopreservation.

After cryopreservation the organ is warmed in an apparatus which is notthe apparatus of this invention.

In preparation for the organ's transplantation into a recipient, theorgan is then reattached to the perfusion apparatus of this invention.

In another embodiment, this invention is directed to a method ofpreparing an organ for transplantation after its cryopreservation andsubsequent warming, comprising:

(a) warming the organ to a temperature which permits reperfusion of theorgan, wherein damage to the organ is minimized;

(b) perfusing the organ with a non-vitrifiable concentration ofcryoprotectant for a time sufficient to permit the approximate osmoticequilibration of the organ to occur; and

(c) perfusing substantially all of the cryoprotectant out of the organwhile concurrently increasing the temperature of the organ to render theorgan suitable for transplantation.

In another embodiment, this invention is directed to a method ofpreparing an organ for transplantation further comprising perfusing theorgan with a reduced concentration of cryoprotectant in combinationwith: a low molecular weight (LMW) "nonpenetrating" osmotic bufferingagent (OBA); or a high molecular weight (HMW) "nonpenetrating" OBA; or acombination of LMW and HMW OBAs which are added and removed in anorchestrated fashion which is appropriate for, and may vary from, organto organ. In the case of the liver, osmotic buffers (OB) do not have tobe used at all. In the case of most other organs, the organ is perfusedwith the appropriate cryoprotectant solution containing a first OBAconcentration for a time sufficient to permit approximate osmoticequilibration of the organ to occur. Substantially all of thecryoprotectant is then washed out (to a final cryoprotectantconcentration of less than 200 millimolar) while decreasing theconcentration of the OBA to a second, nonzero level substantially belowthe first buffering agent concentration level and while concurrentlyincreasing the temperature of the organ. Finally, the organ is perfusedto remove the OBA sufficiently to render the organ suitable fortransplantation.

Exemplifications include the rabbit kidney, the rat liver, and the humankidney.

The apparatus of the invention comprises a computer operated perfusioncircuit containing a plurality of fluid reservoirs, a means for raisingand lowering concentrations and an organ container. A first fluid flowpath is defined as a loop from the plurality of reservoirs to necessarysensors and temperature conditioning means and back to the plurality ofreservoirs. The reservoirs are selectively connectable to the firstfluid flow path. Pump means are interposed in a second fluid flow pathfor pumping fluid from the first fluid flow path to a second fluid flowpath. The organ container is located in this second fluid flow path.Pump means may also be included in the second fluid flow path forpumping fluid from the organ container to one or more of the reservoirsor to waste. One or more sensors are interposed in the fluid flow pathsfor sensing at least one of the concentration, concentrationdifferential, temperature, pressure, and pH of the fluid flowing in thefirst and/or second fluid flow paths. Measuring means are interposed inthe first and second fluid flow paths for measuring concentration andtemperature differences between the upstream and downstream sides, inthe fluid flow direction, of the organ container. The sensor(s) and themeasuring means are connected to a programmable computer for providing acontinuous information stream from the sensor(s) to the computer.Finally, the computer is coupled to the selection means and the pumpmeans to continuously selectively control (a) the flow of fluid fromeach of the reservoirs individually to the fluid flow paths, (b) theflow of fluid from each of the fluid flow paths individually to each ofthe reservoirs, and (c) at least one of the concentration, temperature,pressure and pH of the fluid flowing in the first and/or second fluidflow path, in accordance with a predetermined computer program withoutsubstantial operator intervention.

Additional features of the apparatus of this invention may include aheat exchanger interposed in the first fluid flow path for conditioningthe temperature of fluid flowing in this fluid flow path. A second heatexchanger may be interposed in the second fluid flow path forconditioning the temperature of fluid flowing in the second fluid flowpath.

In describing the apparatus and methods of this invention, many of thevarious aspects of the same have been numbered. This numbering has beendone to create a conceptual organization and structure for thisapplication. This numbering should not be interpreted to necessarilymean or imply that the particular steps in this invention must beperformed in the sequences in which they are presented.

FEATURES AND ADVANTAGES OF THE INVENTION

This invention has a multitude of features and advantages, among themost important of which are the following.

1. It permits control of the concentration of cryoprotectant or anyother fluid or drug in the perfusate of an organ according to a widevariety of predetermined concentration-time histories, more or lessindependently of the flow rate of perfusate through the organ, withprovision for simultaneously varying the concentrations of other drugsor osmotic agents. Step changes in concentration are possible, and it ispossible to bring concentrations effectively to zero.

2. It provides for in-line sensing of concentration, pH, perfusatetemperature, and other parameters so as to avoid the need for sensors inthe perfusate reservoirs and for manual measurements.

3. It permits minimizing differences between the concentration ofcryoprotectant monitored and the concentration of cryoprotectant in theperfusate reservoirs by minimizing the time required for perfusate totravel from the reservoirs to the perfusate sensors and back to thereservoirs.

4. It permits minimizing differences between the concentration ofcryoprotectant monitored and the concentration of cryoprotectantactually perfused into the organ by minimizing the time required for theperfusate to travel from the main fluid circuit to the perfused organ(or superfused tissue).

5. It permits monitoring of the arterio-venous difference incryoprotectant concentration across the organ as an index of the degreeof, or opportunity for, organ equilibration with cryoprotectant.

6. It permits control of the temperature of the organ essentiallyindependently of the flow of perfusate through the organ, and permitsvarying this temperature at will.

7. It permits control of the perfusion pressure, either keeping it fixedor changing it as desired, and, if desired, minimizing pulsation.

8. It protects against perfusion of unmixed solution, air bubbles,particulates, or pathogens into the organ, and avoids inaccessible fluiddead spaces.

9. It interfaces with a computer to control the perfusions, to providereal-time monitoring, display, processing, and recording of the data, tocalibrate the sensors and pumps, and to direct the cleaning,disinfection, and priming of the perfusion circuit and to instruct andalert the operator, when necessary.

10. It is readily capable of perfusing and cryoprotecting organs ofwidely varying size and perfusion requirements, e.g., anything from arat heart to a human liver, and is capable of tissue or cell culturesuperfusion as well.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the overall fluidic circuit diagram of this invention.FIG. 1B shows the construction of the Effluent Distribution Block (EDB)and the means by which the effluent flow is divided to allow sampling bythe Δ R.I. pump 126 in FIG. 1A.

FIGS. 2A-2C show side, top and bottom views, respectively of atwo-chamber gradient former employed as reservoir R1 in this invention.

FIGS. 3A--3C show side, top and bottom views, respectively, of athree-chamber gradient former used as reservoir R3 in this invention.

FIGS. 4A-4C show left, front, and right side views, respectively, of theHeat Exchanger/Bubble Trap/Mixer (HBM) used in this invention; FIG. 4Dshows the basic mixing unit area of the HBM; and FIG. 4E shows a topview of the base of the HBM.

FIG. 5 shows a typical protocol for introducing and removing arelatively dilute vitrification solution. As used in FIG. 5 and in somelater figures the following abbreviations have the following meanings:

pH5 means phase 5;

epH6 and 7 mean the end of phases 6 and 7, respectively;

pH5:250 means that the concentration of LMW OBA during phase 5 was 250millimolar;

epH6:50 and epH7:0 mean that the concentrations of LMW OBA at the end ofphases 6 and 7 were 50 and 0 millimolar, respectively;

Veh. means vehicle.;

EC means Eurocollins solution;

CPA means cryoprotectant agent;

Numbers 1 to 7 within circles designate the 7 phases referred to later;

P/10 means pressure divided by 10;

M means the target molar concentration;

M means the measured molar concentration; and

F means flow in ml/min.

FIG. 6 shows the part of the protocol for the two-step introduction offully concentrated vitrification solution that was carried out insidethe standard perfusion machine.

FIGS. 7A-7D comprise a flow chart of activities for organ cryoprotectantperfusion.

FIG. 8 is a schematic diagram of the details of the two-step coolingtechnique for introducing high concentrations of cryoprotectants.

FIG. 9 shows an apparatus to perfuse kidneys with vitrificationsolutions outside of the standard perfusion machine and at temperaturesin the vicinity of -20° to -30° C.

FIG. 10 shows a typical rat liver perfusion protocol in which neitherHES nor LMW OBAs were used.

FIGS. 11A and 11B comprise a flow chart of the procedure fornon-cryoprotectant perfusions.

FIG. 12 shows the ability of rabbit kidneys transplanted after perfusionwith the vitrifiable solution known as V49 to function as measured bytheir control of serum creatinine.

FIG. 13 shows the effect of cooling to -30° C. on rabbit kidneyspreviously perfused with 7.5M or 8M cyroprotective agents, in comparisonto the results for the non-cooled kidneys exposed to -3° C.

FIG. 14 shows the results of exposing rabbit kidney slices to highconcentrations of cryoprotectant after rather than prior to cooling to-23° C., demonstrating that both the cooling injury and the toxicityassociated with high concentrations are prevented by cooling initiallyin a low (6.1M) concentration.

FIG. 15 shows that cooling injury is also successfully avoided in theintact kidney at 6.1M croprotectant (100% survival, excellent finalcreatinine levels), proving the hypothesis that cooling injury isabolished at low concentrations.

FIG. 16 shows the feasibility of the two-step approach for introducing8M cryoprotectant at -22° C.; the survival rate was 7/8 and thecreatinine levels after two weeks were excellent and identical to thosefor kidneys exposed only to 6.1M cryoprotectant.

FIG. 17 shows the feasibility of using the two-step approach to avoidcooling injury down to -32° C. with 8M cryoprotectant (survivalrate=100%, final creatinine levels identical to those for kidneysexposed only to 6.1M cryoprotectant).

FIG. 18 shows that kidney slices treated with V55 and cooled to -46° C.experience maximum cooling injury, no further injury being apparent whenslices were cooled all the way to the glass transition temperature.

FIG. 19 shows the postoperative serum creatinine levels in an intactkidney that was treated with V55 and cooled to -46° C. with subsequentlife support funtion (survival rate: 1/1 kidneys so treated; finalcreatinine levels: acceptable.)

FIGS. 20A and 20B show data from the perfusion of a human kidney withthe vitrifiable solution known as V55 by the method of this invention.Specifically, FIG. 20A shows successful control of cryoprotectantconcentration. FIG. 20B shows resistance and flow data. The data are allfrom the same 232 gram human kidney. In FIG. 20A, P means pressure in mmHg. In FIG. 20B, resistance is expressed as weight times pressuredivided by flow rate ##EQU1##

FIG. 21 shows cooling data from the same kidney as FIG. 20. The kidneywas cooled after immersion in a 60% w/v mixture of dimethyl sulfoxideand acetamide. These data gave a continuous recording of organ coretemperature from 0° C., which was reached in about 15 minutes, to aboutthe glass transition temperature. The data revealed no evidence of iceformation within the kidney.

FIG. 22 shows loading (ascending portion) and unloading (descendingportion) of a human pediatric kidney with V55 using the method of thisinvention. The solid line was the target V55 concentration while thedotted line was the actual measured V55 concentration in the circuit.Since the cryoprotectant was unloaded from this kidney a cooling curvewas not generated.

FIGS. 23A-23C show viability data for rabbit kidney slices (FIG. 23A),human kidney slices (FIG. 23B) and comparative rabbit:human data (FIG.23C). The human kidney slices showed identical responses to V49 as therabbit slices but showed slightly lower recovery after cooling to -30°C.

DEFINITIONS

In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided. Any terms which are notspecifically defined in this or other sections of this patentapplication have the ordinary meaning they would have when used by oneof skill in the art to which this invention applies at the time of theinvention.

As used herein, "cryopreservation" means the maintaining of theviability of excised tissues or organs by storing them at very lowtemperatures. Cryopreservation is meant to include freezing andvitrification.

As used herein, "vitrification" means solidification of an organ ortissue without freezing it.

As used herein, "cryoprotectant" means a chemical which inhibits icecrystal formation in a tissue or organ when the organ is cooled tosubzero temperatures and results in an increase in viability afterwarming, in comparison to the effect of cooling without thecryoprotectant.

As used herein, all temperatures are in °C. unless otherwise specified.

As used herein, "non-penetrating" means that the great majority ofmolecules of the chemical do not penetrate into the cells of the tissueor organ but instead remain in the extracellular fluid of the tissue ororgan.

As used herein, "osmotic buffering agent (OBA)" means a LMW or HMW"nonpenetrating" extracellular solute which counteracts the osmoticeffects of greater intracellular than extracellular concentrations ofcryoprotectant during the cryoprotectant efflux process.

As used herein, LMW OBAs have relative molecular masses (M_(r)) of 1,000daltons or less. LMW OBAs include, but are not limited to, maltose,potassium and sodium fructose 1,6-diphosphate, potassium and sodiumlactobionate, potassium and sodium glycerophosphate, maltopentose,stachyose, mannitol, sucrose, glucose, maltotriose, sodium and potassiumgluconate, sodium and potassium glucose 6-phosphate, and raffinose. In amore preferred embodiment the LMW OBA is selected from the groupconsisting of mannitol, sucrose and raffinose.

As used herein, HMW OBAs have M_(r) of 1,000 to 500,000 daltons. HMWOBAs include, but are not limited to, hydroxyethyl starch (HES) 450,000daltons and lower M_(r) hydrolysis fragments thereof, especially 1,000to 100,000 dalton fragments), polyvinylpyrrolidone (PVP), potassiumraffinose undecaacetate (>1,000 daltons) and Ficoll (1,000-100,000daltons). In a most preferred embodiment the HMW OBA is HES, 450,000molecular weight.

As used herein, "approximate osmotic equilibration" means that thedifference between the arterial and venous concentrations is less thanabout 50 to 200 mM. (A difference of 200 mM at an arterial concentrationof 4M means that the venous concentration is 95% of the arterialconcentration. A 153 mM difference is equivalent to a 1% w/vconcentration difference for our preferred cryoprotectant formuladescribed below.)

As used herein, "animal" means a mammal including, but not limited to,human beings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT AND BEST MODE

1. L Description of the Perfusion Apparatus

In a preferred embodiment, the apparatus incorporating the principlesand features of this invention is contained in a refrigerated cabinet100 (shown by double dashed lines in FIG. 1A). The refrigerated cabinetcontains two sides, the reservoir/solenoid side and theorgan/refractometer side. The cabinet is faced with double panedtransparent doors each containing approximately 1 inch of insulating air(which can be reduced in pressure and/or humidity if necessary) betweenthe panes to avoid condensation of moisture on the doors and to minimizeheat leak into the cabinet. The organ-side door is split to form a"Dutch door". This allows the upper portion of the organ-side door to beopened and closed to place the organ in the system and to remove theorgan without changing the temperature below the upper portion of thedoor, where the organ container and most other equipment are located.The cabinet may also employ a "Dutch door" on the reservoir side of thecabinet to enable the operator to make any needed adjustments (e.g.,fluid addition to the reservoirs, transfer of upper fluid lines, etc.)without disturbing the cabinet's temperature to an unnecessary degree.

The primary features of the invention and its mode of operation areshown in the fluidic logic schematic of FIG. 1A. All fluid available forcirculation through the system is drawn into the main circuit by acircuit pump 102 through fluid uptake lines U1, U2, U3, or U4 dependingupon the computer-controlled actuation pattern of three-way solenoidvalves S1, S2, and S3. Uptake lines U1-U4 connect either to fluiddelivery lines D1-D4 leading from reservoirs R1-R4, respectively, or tocleaning ports C1-C4, through standard tubing quick disconnects. Byclamping D1-D4 and unplugging them from uptake lines U1-U4, lines U1-U4can be plugged into cleaning ports C1-C4, as indicated by the curvedarrows. While this is presently done manually, it will be appreciated bythose skilled in the relevant arts that appropriate valves, tubing andcontrols could be added to handle most of these tasks automatically.

In the embodiment of the invention as presently constructed, thereservoirs R1-R4 are supported on a thick transparent plastic shelf fromwhich four magnetic stir tables hang which stir the four reservoirs (notshown in FIG. 1). Thorough stirring of R1, R3, and R4 is necessary forproper generation of the desired concentration-time histories. Theon/off states and stir rates of the stir tables are independentlycontrolled by instrumentation located outside the refrigerated cabinet.

Ports C1-C4 lead to sources of sterile (distilled) water, air, anddisinfectant. Solenoid valves S0 and S00 are interposed in the deliverylines for these sources and are arranged to ensure that traces ofdisinfectant do not enter the perfusion system by accident. Solenoid S0controls whether air or fluid will enter the perfusion circuit forcleaning, while solenoid S00 determines whether the fluid selected willbe water or disinfectant. The breakup of the main cleaning line intofour independent channels outside of the cabinet rather than just beforereaching C1-C4 (not so indicated in FIG. 1) ensures that each channel isindependent of the others, i.e., not subject to any meaningfulcross-contamination resulting from diffusion of unpurged solutionbackwards from the fluid uptake lines U1-U4 into the cleaning linesleading to cleaning ports C1-C4.

Distilled water and disinfectant are drawn into the system through asterilizing filter F4, while air is drawn into the system through an airfilter F5. The disinfectant of choice at present is a clinicallyaccepted dialysis machine cold sterilant such as Actril™ (Minntech,Minneapolis, Minn.). The cleaning procedure is to wash the perfusate outof the system with water and then to displace the water with sterilant.Prior to the next perfusion, the sterilant is washed out of the systemwith water and the water is then washed out of the system with air. Thesystem is then primed by displacing the air with appropriate perfusate.The air flush is used to avoid the persistence of any lingering tracesof sterilant dissolved in the rinse water, and to avoid any possibledilution of the priming fluid with water (i.e., to reduce the amount ofpriming fluid needed for displacing water from the system), to allow avisual check of the completeness of priming, and to reduce spillage ofwater in the cabinet when the reservoirs, filters, and organ cassetteare placed into the system after cleaning but before priming. The airpurge can, however, be omitted if desired. The air filter is used toprevent contamination from pathogens in the air, if necessary.

Solenoid valves S9-S12 normally direct fluid to reservoirs R1-R4 or tothe waste line (LW). Reservoirs R1-R4 can also be detached from thesystem by removing recirculation lines RL5-RL8 from reservoirs R1-R4 andplugging them into waste ports W1-W4, respectively (as indicated bycurved arrows), allowing reservoirs R1-R4 to be removed from the systemfor cleaning, sterilizing, and refilling. When reservoirs R1-R4 areremoved, valves S9-S12 direct fluid to waste ports W1-W4. The four wastelines corresponding to waste ports W1-W4 converge to a single commonwaste line LW. A two-way solenoid valve S16 is located on the commonwaste line. When the waste ports are not in use, the common wastedrainage line is blocked by closing valve S16 to prevent any possiblebackflow of waste or pathogens into the sterile cabinet.

The use of this system of uptake lines U1-U4, which are pluggedalternately into reservoir delivery lines D1-D4 or cleaning ports C1-C4,in combination with recirculation lines RL5-RL8, which are pluggedalternately into the reservoir internal return lines (not shown in thefigure) or into waste ports W1-W4, allows complete sterilization of theperfusion circuit. The blunt ends of the uptake lines U1-U4, deliverylines D1-D4, cleaning ports C1-C4 and waste ports W1-W4 may besterilized by swabbing with disinfectant when the tubing is beingtransferred from one alternative position to the other. The tubingtransfer is accomplished while applying digital pressure to the tubingso as to occlude it while making the transfer to prevent fluid leaks andfurther reduce the risk of contamination.

The fluid withdrawn from reservoirs R1-R4 or from ports C1-C4 isdelivered through one of several filters F1, F2, and F3, depending uponthe state of actuation of solenoid valves S4 through S7. These actuationpatterns will be described in more detail below. Experience has shown,however, that a single filter F1 or two filters F1, F1' in parallel willbe adequate for most studies (rendering valves S4-S7 optional, asindicated by broken lines) since virtual step changes in concentrationcan be imposed even when only one or two filters in parallel are presentin the circuit.

It is desirable to minimize the distance between the circuit pump 102head and the solenoids S1-S7 to minimize circuit dead space and deadtime and to minimize the effects of perfusate viscosity. Short distancesand adequate tubing inner diameters are particularly critical for S1-S3to assure adequate fluid withdrawal from R1-R4.

Standard Millipore filters appear (Bedford, Mass.) compatible with ourcryoprotectants. The filters are capable of sterilizing the perfusateand are autoclavable. All filter holders can be removed from the systemfor cleaning and sterilization by means of the quick disconnects shownin FIG. 1A. Vent lines V1-V3 lead to solenoid valves S13-S15, locatedoutside of the refrigerated portion of the cabinet 100. These vent linesare opened and closed under computer control during priming and cleaningof the system to permit air to escape and thereby prevent the filtersfrom becoming blocked by air or damaged. A manual bypass (shown only forthe S13 bypass) is provided for V1-V3 for emergency purging of air fromthe circuit. Obviously, air purges of the system beyond filters F1-F3are not possible if filters F1-F3 are present in the circuit; hencefilters F1-F3 must be removed before beginning the washout of sterilantif an air purge is to be included in that procedure.

In the presently preferred embodiment, a 90 mm diameter filter of 0.22micron pore size is located in each filter holder. This size filter isable to pass enough vitrification solution at -6° C. to permit thesuccessful perfusion of a rabbit kidney, with an overlying 1.2 micronfilter and a coarse prefilter to prevent clogging. The standardconfiguration for the operative version employs two identical filters inparallel. This is necessary to accommodate the flows required for humanorgans and provides a safety factor for any air which may beinadvertently introduced into the arterial fluid, as well as minimizingpressure build-up proximal to the filter. This continuous filtersterilization and resterilization of the perfusate during the perfusioncan serve as a back up for pre-sterilized solutions in case ofcontamination for any reason during the perfusion. (The incidence ofrenal infections has been 0% after literally several hundredperfusions.)

Once the fluid from the selected reservoir has passed through theappropriate filter, it goes through some preliminary temperatureconditioning in a heat exchanger 104 and then travels to a position asclose to the organ as possible, at which point it encounters a "T" typetubing connector T1. The bulk of the flow passively takes the path L1("refractometer loop") that leads to a flow-through process controlrefractometer 106 that measures the index of refraction of the liquidand hence the cryoprotectant concentration. The remainder of the flow isdirected through an organ loop L2 by means of an organ pump 108. Theorgan pump speed is controlled by the computer so as to maintain thedesired organ perfusion pressure despite wide variations in the organ'svascular resistance. By changing the organ pump head and the diameter ofthe tubing going through it, a wide range of flows can be generatedsufficient to perfuse organs of a wide range of sizes: organs as smallas rat hearts to organs as large as human kidneys have been successfullyperfused.

The flow rate delivered by the circuit pump 102, which supplies both therefractometer loop L1 and the organ loop L2, must be high enough to bothexceed the flow rate through the organ at all times and to ensure thatsufficient flow is available for the refractometer 106 and other in-linesensors, generally designated 110, for measuring temperature, pH, andother desired parameters of the perfusate, to permit accuratemeasurements. The flow must also be high enough to minimize the "deadtime" between changes in reservoir concentration and changes in thesensed concentration and other sensed parameters in the refractometerloop as well as to minimize the "dead time" between the reservoir andthe organ. The circuit pump flow is limited by the need to prevent fluidfrom being delivered to the filters at a rate in excess of what thesefilters or the tubing leading to them can pass without failing, as wellas by constraints of heat output and wear and tear on the circuit pumptubing. The speed of the circuit pump is usually not varied during anexperiment and does not therefore usually require computer control,though computer control is available as an option.

After passing through the organ pump 108, the perfusate passes through asecond heat exchanger 112 that finalizes perfusate temperatureconditioning. This is done by adjusting the flow of both cold and warmliquid from cold and warm baths 114, 116, respectively, usingcomputer-controlled pumps (not shown) between heat exchanger 112 andbaths 114 and 116.

The computer is able to vary flow through both the cold path and thewarm path so as to adjust perfusate temperature in the arterial line andtherefore also in the effluent of the organ. The arterial and effluenttemperatures provide an indication of the actual organ temperature. Bycontrolling the flow rate of cold and warm bath fluid, organ temperaturecan be adjusted independently of organ flow, provided flow is not closeto zero. Experience has shown that arterial and venous temperatures atleast as cold as -6° C. and at least as high as 25° C. can be achievedwith this invention. Generalized cabinet cooling is not an alternativeto the heat exchange system shown for subzero perfusions because coolingof the cabinets to subzero temperatures will cause freezing of the moredilute solutions in the tubing lines. Specific jacketing and cooling ofthe organ container is of particular theoretical value, however, and mayoptionally be included.

The temperature-conditioned perfusate is then debubbled and mixed in abubble trap/mixer 120 just before entering an organ container 122.Arterial and venous temperature probes, generally designated "T" in FIG.1A, penetrate the wall of organ container 122 through simple holes.Pressure and, optionally, temperature is sensed in the bubble trap.Although shown separately in the drawing for ease of understanding, thebubble trap and mixer 120 are in fact an integral part of the heatexchanger 112, so heat exchange continues to be controlled whiledebubbling and mixing are accomplished. Experience has shown that mixingwas important due to the tendency for layering of dilute solutions onmore concentrated, denser solutions. Details as to the specificconstruction of the heat exchanger/bubble trap/mixer (HBM) are describedbelow.

Under normal circumstances, the cooling fluid effluent from this secondheat exchanger 112 is used to cool the perfusate passing through thepreliminary heat exchanger 104. This cooling fluid then travels to asolenoid holding block 118 physically containing solenoids S1-S12, so asto draw off waste heat from these solenoids before returning to the coldbath.

The holding block 118 currently consists of a large aluminum block (butmay be either metal or plastic) perforated with cylindrical holes ofsufficient diameter to closely match the outside diameters of the heldsolenoids. The solenoids are inserted such that the base, containing thefluid inlets and outlets, faces the operator and the head, from whichthe electrical leads penetrate into or through the holding block. Thesolenoid holding block is equipped with an internal fluid path fordrawing off waste heat from the solenoids. Feet are provided to positionthe holding block, prevent it from moving, and protect the fluid inletand outlet ports when the holding block is removed from the cabinet. Theblock is positioned behind and above the reservoirs in the refrigeratedcabinet so that the solenoid inlets and outlets and their connections tothe reservoirs are always readily visible.

The solenoids are preferably 3-7 watt (or less) piston type 3-waysolenoids of minimal internal fluid volume having orifices on the orderof 0.156 inches or more and Cv values ≧0.16 (e.g., Model 648T033solenoids from Neptune Research, Maplewood, N.J.) while resistingpressures of up to 500 mm Hg or so. The inventors presently preferNeptune Research (supra), 3-watt solenoids fitted with RC droppingcircuits to reduce heat generation after activation. Solenoids having1/16 inch orifices and Cv values of 0.01 to 0.03, e.g., Valcor's Model20-2-3 (Valcor Scientific, Springfield, N.J.) are not fully satisfactorydue to the high viscosity of the solutions used for cryopreservation(causing difficulty aspirating viscous fluid through S1-S3), the highflows desired for controlling dead times and for perfusing largerorgans, the possibility of clogging, and the buildup of pressure betweenthe circuit pump and S8-S12. The detailed actuation pattern of thesolenoids is described below. The solenoids inside the refrigeratedcabinet that are not held in the solenoid block, SR1, SR31 and SR32, aredescribed in more detail below.

An effluent distribution block (EDB) 124 (FIG. 1A) is connected to theoutput side of the organ container 122. The EDB is designed so that asmall amount of effluent is always present at the bottom of the block.This residual fluid is withdrawn by the two-channel "delta R.I. pump"126 and sent to the differential refractometer ("delta R.I. meter") 130where its refractive index (a measure of concentration) is compared tothat of the perfusate from refractometer loop L1 (pumped at the samerate as the venous effluent sample) and a difference signal generatedand sent to the computer. Since the fluid in the refractometer loop L1will approximate the concentration of the fluid entering the artery ofthe organ, the delta R.I. output provides an estimate of thearterio-venous concentration gradient across the organ. When thisgradient is large (in either the positive or negative direction), theorgan is far from equilibrium. When the gradient is zero, the organ isat least largely in osmotic equilibrium with the perfusate. Thenonlinear baseline resulting from this unorthodox use of thedifferential refractometer is accounted for in the software for runningthe perfusion program.

All effluent from the organ (together with the arterial fluid sampled bythe delta R.I. pump) is ultimately collected by the recirculation pump128 and sent to solenoid S8, which controls whether the effluent isrecirculated to the reservoirs or discarded. Effluent to be returned toa reservoir is combined with the fluid flowing through the refractometerloop L1 at a T connection T2. As noted above, return to the correctreservoir is then controlled by the actuation of solenoids S9 throughS12.

The recirculation pump 128, like the circuit pump 102, need not requireflow adjustment. It is normally set to a rate sufficient to exceed themaximum flow through the organ pump 108. Since the output of therecirculation pump exceeds that of the organ pump, air is continuallyintroduced into the tubing leading to solenoid S8 and usually to thereservoirs R1-R4. Provisions to prevent excessive bubbling of thereservoirs as a result of this are described below.

Although the delta R.I. pump speed can be changed, it is usually keptconstant throughout an experiment. In the presently operative version,it has not been under computer control, but computer control would be adesirable option in some cases. The delta R.I. pump employs very smalldiameter polyethylene tubing to reduce delays in fluid transit time.This small tubing is particularly important because the flow ratethrough the delta R.I. circuit is limited by the lowest flow ratethrough the organ, which may be small, and by the limited size of thefluid paths in commercially available differential refractometers.

The return of the differential refractometer output to the organeffluent line is proximal to the effluent recirculation pump. Thisplacement rather than placement distal to the pump ensures a steady flowthrough the differential refractometer, whereas distal placement mayprevent or alter differential refractometer flow by virtue of a higherexit pressure.

An important element of the fluidic circuit is the gradient pump 132connected to the circuit by a line P1 (FIG. 1A). The function of thegradient pump is to allow for gradual changes in concentration withinthe appropriate reservoirs within the cabinet. The method by which thisis accomplished will be described below. The placement of the line P1 tothe gradient pump at T3A, just after the point of joining of therefractometer loop L1 and the organ loop L2, presents one option forensuring the removal of some of the air introduced by the organ effluentrecirculation pump 128 and therefore helps reduce bubbling of thereservoir fluid.

A better option, however, and the one presently used, is to draw no airinto line P1. This is accomplished by connecting P1 at point T3B andresults in fully controlled concentration-time histories. The bubblingproblem is then overcome by continuously regulating the speed of therecirculation pump 128 to be just slightly in excess of the combinedflows of the organ pump 108 and the delta R.I. pump 126 so as tointroduce little air. Attaching the recirculation output of S8 directlyto P1 without regulating the speed of pump 128 results in degradedconcentration control and is not recommended.

The present operative version of the embodiment of the invention usessilastic tubing of 1/8 inch diameter throughout the system, which issufficient to accommodate the needed flows and is preferred. Silastic iscompatible with Actril™ (Minntech, Minneapolis, Minn.) cold sterilant,is translucent (important for visualizing flow to detect problems andfor observing any signs of microbial growth), is impervious to commoncryoprotective agents such as dimethyl sulfoxide, and is soft enough tobe easily manipulated. However, silastic tubing should not be used incircuits coming into contact with silicone cooling fluids, which swelland weaken silastic tubing. In addition, C-Flex® tubing (Cole PalmerInstrument Co., Chicago, Ill.) should be used in the pump heads due toits greater strength (silastic tubing undergoes spallation) and greaterflexibility when cold.

Reservoir R1 is constructed as a gradient former (FIG. 2). Essentiallythe gradient former consists of two concentric cylinders, an outercylinder 200 and an inner cylinder 201. A fluid path 205 allows fluid toflow from the outer cylinder 200 to the inner cylinder 201 under theinfluence of gravity in response to a reduction of volume in the innercylinder. The concentric orientation of the fluid compartments is veryspace efficient. The fluid delivery line 204 corresponds to the line D1of FIG. 1A. The unit shown is a modification of a commercially availablegradient former. The necessary modifications for use with this inventionare as follows.

1) The stopcock normally used to control flow from the outer cylinder tothe inner cylinder in the commercial device is replaced by a pinch-typetwo-way (on/off) solenoid valve 202 (currently, a Bio-Chem Valve Corp.model 100P2WNC, East Hanover, N.J.) (FIG. 2C). A pinch-type valve ispreferable for this application to a piston-type valve because of thesmall pressure difference available to drive fluid flow and theconsequent need for a large working diameter fluid path 202b. It is alsopreferable for easy removal from its tubing 202b when the reservoir isto be removed from the cabinet for cleaning, leaving the solenoidbehind. The base of the gradient former has been modified, at 203, tomake room for the solenoid and to support it on a platform. Platform 203is equipped with a vertical metal post 203b. Solenoid 202 is lashed tothis post with a rubber band so as to keep the solenoid orientedcorrectly. The solenoid is located a sufficient distance from thereservoir to avoid excessive heating of the reservoir fluids.

2) The diameter of the fluid path 205 from the outer cylinder 200 to theinner cylinder 201 has been enlarged to permit flow at an adequate rateof the viscous solutions required for organ cryopreservation. An innerdiameter of 1/8 to 3/16 inch is adequate.

3) A lid 206 has been provided (FIG. 2B). The lid has an outer overhang207 that prevents the lid from moving from side to side after it isplaced on the cylinder as well as concentric grooves into which thewells of 200 and 201 fit. The lid has built-in outer and inner fillingfunnels 208a and 208b with removable lids, and a recirculation port 209.

4) Funnels 208a and 208b extend into respective internal fill tubes 210aand 210b. The internal fill tubes are preferably rigid hollow rodslocated next to the wall of the inner and outer cylinders and areperforated at 1-2 cm intervals with holes 211a and 211b, respectively,which are approximately 3 mm in diameter. The function of the fill tubesis to reduce the creation of bubbles as recirculating fluid impacts thesurface of the liquid in the reservoir. The purpose of the perforationsis to enable air to escape from the tube through the perforations so asnot to force air to the bottom of the reservoir to form bubbles. Thesefunctions are particularly important in perfusates containing protein,which tend to stabilize bubbles.

5) A fill mark has been provided to enable the reservoir to be filledreproducibly to the same, predetermined volume. The operator canestablish his/her own fill mark depending upon the details of theapplication. The gradient formers may have approximate graduations(horizontal lines on both the inner and outer cylinders, aligned so asto permit avoidance of parallax error in reading the liquid level ineither cylinder) spaced approximately 0.5 cm apart for a 2 litergradient former. These graduations are also important for establishingslight, deliberate mismatches in liquid level between inner and outercylinders, which are necessary to prevent premature mixing of solutionsof widely differing densities, such as cryoprotectant-free perfusate andvitrification solution. They also permit a rough quantitative check bythe operator on the progress of the gradient as represented on thecomputer screen.

6) The plastic composition of commercially available gradient formersmay create problems for certain types of cryoprotectant, which couldconceivably attack the plastic. It is therefore preferred to usereservoirs made of transparent material (e.g., glass, plexiglass or thelike) that is compatible with the cryoprotectant chemicals or usereservoirs whose surfaces have been siliconized or otherwise treated toprevent the attack. In the inventors' experience, acrylic has been foundto be an acceptable material.

7) The reservoir R1 contains a stir bar 212. The stir bar is housed in ajacket 213 attached to a freely spinning vertical pin 214 extending tothe stir bar from the lid of the reservoir to prevent the jacket, andhence the stir bar, from moving laterally. This change is necessary tomake sure chattering, and therefore poor mixing, does not occur whilethe perfusion machine is unattended. Support from above rather thanbelow prevents unnecessary perfusate frictional heating and wear andtear to the floor of the reservoir.

Reservoir R3 is also constructed as a gradient former. The details ofreservoir R3 are shown in FIG. 3. Reservoir R3 contains an outercompartment 315 (R3₃), an inner compartment 318 (R3₁), and a thirdintermediate compartment 316 (R3₂). Intermediate compartment 316 isconnected to inner compartment 318 through a fluid conduit 320controlled by a solenoid 317 (SR3₁). Compartment 316 also connects toouter compartment 315 by a fluid conduit 321 controlled by a solenoid319 (SR3₂). The use of an outer compartment is necessary whenconcentration is being reduced to zero or nearly zero, for reasons notedbelow in the discussion of the function of the gradient pump and theaction of the gradient formers. The use of an outer compartment isgreatly preferred compared to a middle compartment having a largervolume of fluid (and no outer compartment) because simply increasing thevolume of fluid in the middle compartment would cause the concentrationprofile resulting from a constant gradient pump 132 flow rate to becomenon-linear. Control of concentration-time history would then become morecomplicated. More importantly, an excessive amount of fluid in themiddle compartment would be required to approach a zero concentration inthe circuit compared to the amount of fluid required in the outercompartment after virtual emptying of the inner and middle compartments.

Automated use of reservoir R3 poses some problems which are successfullyaddressed in part by software and in part by the specific constructionof R3. Specifically, actuation of solenoid SR3₂ allows fluid in theouter compartment (R3₃) to flow first into the middle compartment (R3₂)and from this compartment to the inner cylinder (R3₁). This is becausethe pressure head present between R3₃ and R3₂ is large when R3₁ and R3₂are nearly empty, which occurs when SR3₂ is activated. At this point,R3₃ is still full. This large pressure head causes fluid to flow toorapidly into R3₁ if R3₃ is connected directly to R3₁ rather than usingR3₂ as a buffer between R3₃ and R3₁. By adjusting the level of R3₃, theflow can also be partially controlled. But even with these twoprecautions, further control of flow is required by using an appropriateduty cycle for SR3₂. The flow to R3₁ should be slow at first and moreand more rapid as the concentration is brought closer and closer tozero, whereas passive flow under the influence of gravity will always befastest at first and slowest at the end unless the flow is metered bythe sort of tailored duty cycle currently being imposed on SR3₂.

The other modifications to R3 resemble those of R1.

Reservoir R4 is a gradient former constructed in the same manner as R1.

The purpose of the gradient pump 132 is to remove some of therecirculating fluid from the circuit. This removal of fluid causes theflow rate of fluid back to the reservoir of origin to be less than theflow rate of fluid from this reservoir to the circuit. This causes thelevel in the inner cylinder of the reservoir (R1, R3, or R4) to go down.This lowering of inner cylinder fluid level in turn causes the fluid inthe outer or middle compartments to flow into the inner cylinder to keepthe two levels similar. Thus the two dissimilar concentrations in thetwo cylinders are mixed in the inner cylinder, generating theconcentration gradient which is then sent to the rest of the circuit.This is the manner in which the gradient pump effects the desiredgradual changes in concentration which reach the organ and therefractometers. Any necessary adjustments to the gradient pump speed aremade by the computer.

The principle involved is that of an ordinary linear gradient former inwhich the portion of the circuit external to the gradient former can beregarded, to a first approximation, as extra volume in the innercylinder. Withdrawal and discard of fluid from the inner cylinder at aconstant rate will result in a linear molar concentration change withtime despite the presence of the rest of the circuit and therecirculation of fluid back to the reservoir. However, unlike anordinary gradient former, the concentration of fluid leaving thegradient former at the moment the volume in the gradient former becomeszero will not be equal to the concentration of fluid in the outer (ormiddle) cylinder of the gradient former. Therefore, in order to approacha concentration of zero during cryoprotectant washout using an ordinarytwo-compartment gradient former, it is necessary to add additional fluidto the outer cylinder while continuing to discard fluid from the innercylinder normally. This is why R3 has been modified to have a thirdcompartment. The extra fluid required to continue cryoprotectant washoutis added from this third compartment by the computer more accuratelythan a human operator could accomplish this task manually. Duringintroduction of cryoprotectant, on the other hand, the desired finalconcentration can always be reached by using a concentration in theouter compartment which significantly exceeds the final concentrationdesired in the circuit at the end of the gradient. Since the currentmethod involves an upward step change in concentration (see below), itis convenient to fill R1's outer compartment with the same fluid used inR2.

The HBM heat exchange system is shown in detail in FIGS. 4A-E.

Perfusate enters the HBM through an entry port 403, travels through acentral channel 400, and leaves the HBM via an outlet port 406. Oneither side of the central perfusate path are separate chambers forregulating temperature. The two innermost temperature control chambers401 (one on each side of the perfusate path) are used for thecirculation of coolant, while the outer chambers 402 are a pathway forthe flow of room temperature fluid for offsetting the coolant. (Forspecialized applications involving, for example, normothermic perfusion,these pathways can be reversed.)

The direction of cold fluid flow is optional. Adequate temperaturecontrol has been found when all fluids (perfusate, coolant, and warmingfluid) flow in the same direction (uphill) despite the lack ofcountercurrent heat exchange. This mode allows the avoidance of air orcarbon dioxide accumulation in the outer chambers.

Perfusate enters the bottom of the HBM unit through inlet 403 andtravels upward in a zig zag pattern. It emerges into a small upperreservoir which has an air space above: this is the bubble trap area404. Perfusate then travels beneath the bubble trap and goes through aperfusate mixing area 405 before finally traveling onward to thearterial outlet.

The inlets for cold 407 and warm 408 fluid are each split into twochannels within the base of the unit. The outlets 410, 411 for warm andcold fluid, respectively, each receive fluid collected from two channelssuch that each channel of the same kind (i.e., each cold channel or eachwarm channel) is the same length and nominally experiences the samepressure difference from start to finish, so that flow rate through eachlike channel should be approximately equal.

All of the cold and warm fluid pathways include a length of flexibletubing 412 at the rear of the unit. These tubing segments serve twopurposes. First, by introducing an air gap between the four channels,heat exchange between them is minimized. This is particularly desirablewhen all of the cold and warm fluid is flowing in the direction oppositeto that of perfusate flow (i.e., in orthograde direction) and has notalready undergone heat exchange with the perfusate. Second, each tubecan be clamped. In this way, if by chance one cold channel or one warmchannel should take all of the cold or warm fluid delivered while theother channel "airlocks", this situation can be corrected by clampingthe channel receiving all of the flow and purging the air out of theinactive channel, bringing each channel into full function and equalflow.

Because in the orthograde mode the temperature conditioning fluid entersthe heat exchanging portion of the unit at the top and exits at thebottom, it is necessary upon installation to run the cold and hot pumpsin retrograde direction in order to purge all air out of the cold andwarm channels. This is best accomplished if the cold and warm tubingleading to and from the bath is no more than about 1/8 inch in internaldiameter, since at this diameter fluid flow will displace air from thetubing rather than allowing it to flow uphill in a direction opposite tothe direction of fluid flow or otherwise to remain unpurged in variousparts of the tubing. Thus, when the pump direction is reversed againfrom retrograde to orthograde, no air will be present in the tubing andnone will be trapped in the heat exchange chambers of the unit.

In addition to serving a heat exchange function, the zig zag pattern isalso designed to force mixing of previously perfused dense perfusate or,when perfusate density is rising rather than falling, to purge the lessdense perfusate from the perfusate path.

As the perfusate emerges from the zig zag heat exchange area, it entersthe bubble trap 404 at trap entry area 418. Perfusate exits the bubbletrap through exit region 419. The zig zag pattern, in fact, is alsodesigned to allow any air bubbles to exit the heat exchange area and toemerge into the bubble trap area. The bubble trap area is designed tohave the following features.

1. Its volume is sufficiently large to reduce the pulsatile action ofthe perfusion pump to a minimum by distributing the shock of each strokeover a relatively large air volume. This simplifies pressure control andmeasurement and may be less damaging to the organ.

2. Its volume is sufficiently low to minimize the liquid volume presentin the trap and thereby to minimize the dead time and dead volumebetween the organ pump and the organ itself. A minimal volume is alsodesirable to minimize layering of more dilute perfusate over more denseperfusate.

3. A pressure sensing port 413 is provided. Port 413 has no fluidconnection to the perfusate, thus eliminating a "blind alley" in whichfluid cannot be mixed properly or in which disinfectant might fail topenetrate or might be trapped. Both an electronic pressure transducer(to provide a signal to the computer) and a sphygmomanometer gauge (forcalibration and visual checking) are used.

4. The lid 414 of the trap is removable for cleaning.

5. A vent port 416 is provided which is used to adjust fluid level inthe trap so as to make it the minimum required to serve the bubble trapfunction and to maximize pressure wave damping. The tubing from thisvent leads to the outside of the cabinet, permitting adjustments to bemade without opening the cabinet door.

6. A third port 417 is provided through the bubble trap lid to permitthe injection of drugs, vascular labeling materials, fixative, etc.

7. The walls of the bubble trap are angled near the trap entry and exitpoints 418, 419, respectively, to produce a certain amount of mixing ofthe perfusate both as it enters and as it leaves the trap, and to breakup and minimize the volume of layers of dilute perfusate overlying moredense perfusate.

8. The option exists of introducing probes, such as a temperature probevia one of the ports in the trap lid to make measurements in theperfusate without permanent embedding of the sensor: the port consistsof flexible tubing attached to a plastic threaded fitting. A probe canbe freely admitted or withdrawn and the tubing clamped with hemostats oran equivalent clamp to effect a pressure-tight seal. This simplifiesremoval and reinstallation of the HBM when it must be cleaned and allowsflexibility in probe selection and the opportunity of using the probefor other measurements elsewhere.

After leaving the bubble trap, the perfusate descends to a mixing area405 (see FIG. 4D). The basic unit of the 3-unit mixing path is a narrowhorizontal entry area HE emerging into a "wide" basal area BA whichrises to an area of flow restriction FR and ends in a descent D to thenext horizontal entry area. Fluid entering HE is forced through anopening too small to support much layering of low density fluid on topof high density fluid, especially considering the right angle turnrequired just before HE. Fluid flowing into BA may, if less dense, riseimmediately upward toward FR. If more dense, it may be driven into thewall and rise upward along this wall. Upon encountering FR, however, thedenser liquid will be accelerated toward the less dense liquid risingdirectly from HE, creating turbulence and mixing. If BA fills with denseperfusate, again the speed of the fluid flowing directly upwards at FRshould cause the dense liquid to mix with any low density fluid layeredabove FR. Furthermore, the narrow descending path D should draw layeredliquid down the angle along with denser liquid, again preventingstagnant layers from persisting. In practice, three such mixing unitsaligned in series as shown in FIG. 4B are sufficient to mix initiallyvery poorly mixed perfusate, which is encountered frequently in thecourse of abruptly raising or lowering cryoprotectant concentration. Onefinal function of the mixing units is to serve as a trap for any smallbubbles which for any reason are not removed in the bubble trap area.(Bubbles in the mixing area are, however, easily purged by the operatorprior to initiation of organ perfusion.)

After leaving the mixing region, the perfusate descends to an outletport 406 leading directly to the organ. The path from the final mixingunit to port 406 is deliberately created at an angle to the horizontalin order to provide one last chance of stopping any bubbles fromreaching the organ, since in order to reach the organ a bubble in thispathway would have to flow downhill, contrary to its tendency to flowuphill.

The mixing area and subsequent areas are purged of air by occluding theoutlet tubing affixed to port 406 with the vent open until approximately1/2 inch of fluid has accumulated in the bubble trap. The vent is thenclosed until the pressure has reached about 60-120 mmHg. Finally, fluidis once again allowed to flow freely through port 406. The jet of fluidthrough the mixing area and out port 406 sweeps all air out of the fluidpath from the bubble trap to port 406. If some air persists, it can beremoved by repeating the process. After air has been purged, the vent isopened to allow unnecessary fluid in the bubble trap to exit the trapunder the influence of gravity, reaching a final depth of about 1/8inch. A final depth of 1/8 inch cannot be set before purging the line ofair because insufficient volume exists to avoid refilling the mixingarea with air from the bubble trap during the purging process.

The HBM is designed to require removal for cleaning only infrequently.Disinfection and removal of disinfectant from the bubble trap area iseffected automatically but presently requires some operator attentionafterwards to ensure that all uppermost exposed surfaces are disinfectedand later washed free of disinfectant without contaminating the outlettubes. The option exists of arranging the outlet tubes at 413, 416, and417 in such a way that, with specific solenoids attached to them, theycould be individually purged with water, disinfectant, and air underautomated control, thus relieving the operator of the need for diligencein cleaning the bubble trap.

After the perfusate exits the HBM unit through port 406, it enters theorgan in the organ container 122 (FIG. 1). In the preferred embodiment,the organ container comprises a rectangular box with a hinged lid, lidstop, lid handle, sloped floor, specially sloped feet, arterial andvenous thermocouple inlets, perfusate inlet, and effluent outlet in thefoot opposite the inlet. The slope of the floor is downward in both theright to left and the back to front directions to ensure that all fluidruns to the foot outlet with very little fluid accumulation anywhere inthe container. One needle probe is inserted directly through the wall ofthe arterial line. A second probe is placed directly in the stream offluid emerging from the organ. In typical results, the arterial andvenous temperatures differ by only tenths of a degree, but both areuseful for quality control. The organ container may employ a soft meshsupport for the organ similar to that used in the MOX-100 DCM™ organcassette (Waters Instruments Inc., Rochester, Minn.) or the organ can beplaced directly on the floor of the organ container or on a speciallydesigned independent and removable support. The latter option ispreferred and is presently in use.

The organ container 122 and the organ pump 108 are placed in maximumproximity to reduce dead times and dead volumes between the two, and thetubing leading from the organ pump to the organ container is chosen tobe as small in inner diameter as possible for the same reason.

Most perfusate does not go through the organ loop L2 as described abovebut travels instead from the filters to the in-line analog refractometer106. The presently preferred embodiment of the invention uses a modifiedcommercially available refractometer from Anacon Inc. (Burlington,Mass.). In particular, small diameter tubing inlet and outlets are usedrather than the very large standard Anacon pipe fittings.

The modification of the refractometer sensing head appropriate for thefinal invention could also contain the following additional changes fromthe ordinarily available Anacon unit.

1. The internal volume of the fluid path could be further minimized.

2. Presently, it is necessary to purge the air space of the unit with aslow flow of dry nitrogen gas to prevent condensation of moisture due tothe low temperatures and high humidities prevailing in the cabinet. In amodified version, the electronics area of the sensing device could behermetically sealed with some desiccant inside to eliminate the need fora nitrogen purge.

3. The present unit must be oriented with the fluid flow direction beingvertical and upwards. However, the unit is not built to be used in thisorientation, and body changes could be made to adapt the unit's shape tothis orientation.

The invention allows the operator to access reservoirs in any sequenceand to otherwise custom-design the process which may be of interest. Theoperator is even free to switch solenoid positions depending on what hemay want to do. Nevertheless, the following nominal applicationillustrates the actuation patterns required to deliver fluid from and toeach individual reservoir and filter. It also illustrates the "standardprotocols" for organ cryoprotectant perfusion and for cleaning of thesystem which the system was designed primarily to carry out.

Solenoid S1 admits fluid from R1 when off, or from R2 when activated.Solenoid S2 is open to R3 when not energized, or to R4 when energized.The output of S1 and S2 is to S3, which accepts fluid from S1 (that is,from R1 or R2) when in the resting state and which accepts fluid from S2(i.e., from R3 or R4) when activated. The common outlet for S3 (alwaysopen) leads to the circuit pump 102, which then withdraws fluid from thesolenoid-selected reservoir.

If differential filters are to be included, then the output of thecircuit pump 102 is to S4's common port (always open). When S4 is notenergized, its output is directed to filter F1. The return from filterF1 returns to the normally open port of S5 and exits through the S5common outlet to the refractometer loop L1 and the organ loop L2. If, onthe other hand, S4 is energized, then its output is directed to thecommon inlet port of S6. When S6 is in the resting state, its output isdirected to filter F2, and the return from filter F2 enters S7 throughits normally open port. The output from S7 travels to the normallyclosed port of S5, which must be energized to accept this output. Oncefluid has entered S5, it flows out the S5 common outlet to therefractometer loop and the organ loop. Finally, if S4 is energized andS6 is also energized, fluid will be directed through both of thesevalves and will reach filter F3. The return from filter F3 occurs viathe energized S7 and the energized S5 solenoids and goes to the twoloops L1 and L2 as above. As noted earlier, the use of filters F2 and F3and therefore of solenoids S4, S5, S6, and S7 is optional and will beuseful primarily when very abrupt changes from one solution to anotherare required, or when particularly heavy particulate contaminates mustbe removed.

Effluent from the organ eventually returns to S8. If S8 is activated,the fluid is discarded. If S8 is not activated, the fluid is directedfrom S8 to combine with fluid from the refractometer loop and isreturned to a desired reservoir.

Fluid traveling through the refractometer loop travels successively tosolenoids S9, S10, S11, and S12 and then to the waste line if none ofthese solenoids are energized. Energizing S9 diverts flow to the R1recirculation line. S10's activation (in the absence of activation ofS9) diverts flow to R2. Similarly, selective activation of S11 or S12will, respectively, recirculate fluid to R3 or R4.

There are two basic processes of solenoid-actuated fluid control, onefor actual perfusions and one for system cleaning and priming. Theperfusion process typically proceeds from R1 through R4 whereas primingmust occur in the reverse order to load the fluid uptake and fluidrecirculation lines for reservoirs R2-R4, particularly if filters 172and F3 and their associated lines are used, leaving the circuit primedwith fluid from (typically) R1 (or C1) at the end of the priming (orcleaning) process. The typical sequence of solenoid activations requiredto prime the complete system (or to clean it) is listed in tabular formbelow.

Solenoid Control Sequence For Standardized Rinsing/Priming

The conditions of the solenoid control processes are set forth in Tables1 and 2. The uses of these control processes are to: replace perfusatewith filter-sterilized H₂ O at the end of the process; replace cleaningH₂ O with chemical sterilant between perfusions; remove disinfectantusing filter-sterilized distilled H₂ O; remove water using air; removeair using reservoir fluid, i.e. prime the system.

When only F1 (not F2 or F3) is present, priming (and cleaning) mayproceed in any order of reservoirs, provided, in the case of priming,that the final reservoir corresponds to the first reservoir used for thesubsequent perfusion. Applicants now use a procedure involving momentaryaspiration from R2, then R3, then R4, then R1, taking just enough timeto prime U2, U3, U4, and U1, respectively, followed by computer/userinteractive activation of S12, S11, S10, and S9 to allow manual fillingof RL8, RL7, RL6, and RL5 by syringe with retrograde exhaust via P1,because this procedure saves large quantities of perfusate and is fast.

The standard process of solenoid actuation for withdrawing fluid fromR1-R4 and for creating gradients for a normal perfusion is as follows(assuming (1) use of optional filters F2 and F3, (2) straightforward ortypical use of the gradient-controlling solenoids, and (3) the existenceof a gradient former as R2). The staged completion of a closed circuitupon going from one reservoir to another is to avoid recirculatingsolution of undesired composition to the new reservoir before itscontents have displaced the previous solution from the circuit. If thereis no problem with recirculating the previous solution, the precautionof delayed recirculation can be dropped.

                                      TABLE 1                                     __________________________________________________________________________    Sub-task Accomplished                                                                           Solenoid # (+ = Energized)                                  00*0*             1 2 3 4 5 6 7 8 9 10                                                                              11                                                                              12                                                                              13                                  __________________________________________________________________________    1. Deliver fluid from R4 through F1                                                             - + + - - - - - - - - - **                                  2. Perfuse R4 recirculation tubing to W4                                                        - + + - - - - - - - - + -                                   3. Deliver from R3 through F3                                                                   - - + + + + + - - - - - **                                  4. Perfuse R3 recirculation tubing to W3                                                        - - + + + + + - - - + - -                                   5. R2, F2         + - - + + - - - - - - - **                                  6. R2 recirculation tubing to W2                                                                + - - + + - - - - + - - -                                   7. R1, F1         - - - - - - - - - - - - **                                  8. R1 recirculation tubing to W1                                                                - - - - - - - - + - - - -                                   9. Organ loop discard tubing***                                                                 - - - - - - - + + - - - -                                   __________________________________________________________________________     *If the sequence above is to be done with reservoir fluid, S0 and SS00        will be off. S0 and S00 will also be off if the sequence above is to be       done with water, and the cleaning ports C1-C4 will be connected to uptake     lines U1-U4. If the sequence above is to be done with disinfectant, S0        will be off and S00 will be on. If the sequence is to be done with air, S     will be on and S00 will be off.                                               **S13 (and, optionally, S14 and S15), the filter vent solenoid(s), will b     on for a portion of this step and off for the remainder of this step: it      will be on just long enough to purge air from the line (usually 60 sec. o     step 1 and 30 sec on each of the remaining steps for which the ** notatio     is used). This can be programmed not to happen if the filters are not         present in the system.                                                        ***this step is omitted when priming the system.                              Note:                                                                         Water control solenoid S16 is on (waste tube open for disposal of fluid t     waste) for steps 2, 4, 6, 8, and 9 but off for all other steps.          

                                      TABLE 2                                     __________________________________________________________________________    Solenoid Control Sequence For Standard Perfusion                              Sub-Task Accomplished                                                                            Solenoid # (+ = Energized)                                 00*0*              1 2 3 4 5 6 7 8 9 10                                                                              11                                                                              12                                   __________________________________________________________________________      Initial recirculation to R1                                                                    - - - - - - - - + - - -                                      R1 gradient      Same as 1, but activate SR1                                  From R2 just to F1, no recirculation**                                                         + - - - - - - + - - - -                                      Deliver R2 first solution through F2,                                                          + - - + + - - + - - - -                                      no recirculation                                                              Recirculate R2 solution except                                                                 + - - + + - - + - + - -                                      from organ                                                                    Recirculate all R2 solution                                                                    + - - + + - - - - + - -                                      Run a gradient from reservoir R2‡                                                   Same as 6, but activate SR2                                  Perfuse from R3 just to S6/F2**                                                                - - + + + - - + - - - -                                      Perfuse from R3 to F3, circuit                                                                 - - + + + + + + - - - -                                      open                                                                        10.                                                                             Recirculate to R3 through F3, circuit                                                          - - + + + + + + - - + -                                      partially open                                                                Recirculate all R3 fluid                                                                       - - + + + + + - - - + -                                      Run first part of R3 gradient                                                                  Same as 11, but activate SR31                                Run Second part R3 gradient***                                                                 Same as 11, plus SR31 and SR32                               Open circuit, perfuse from R4                                                                  - + + + + + + + - - - -                                      through F3                                                                    Recirculate to R4 except from                                                                  - + + + + + + + - - - +                                      organ                                                                         Recirculate from both loops to R4                                                              - + + + + + + - - - - +                                      Run R4 gradient  Same as 16, but activate SR4                               __________________________________________________________________________     *For normal perfusions, solenoids S0, S00, and S13-S16 will always be         nonactuated.                                                                  **This step prevents fluid from the previous reservoir, which is initiall     present in the line between the new reservoir and the filter that had bee     previously equilibrated with fluid from the new reservoir, from               contaminating the previously equilibrated (new) filter.                       ***As noted in the discussion, SR32 activation must follow a duty cycle       initially, ending in permanent activation of SR32 until end of use of R3.     The duty cycle involves switching back and forth between solenoid pattern     12 and 13 as dictated by the duty cycle requirements.                         ‡Step 7 is optional.                                          

The number of reservoirs could be less than or greater than the numberspecified here, with corresponding changes in solenoid number.Furthermore, the number of layers of R1-R4 need not conform to thedescriptions given above. The limits would be one reservoir at the leastand perhaps eight reservoirs at the maximum, in which any reservoircould have from one to four compartments. The upper limits are basedpartly on volume and crowding constraints and partly on theimprobability of any procedure complex enough to require more than 8reservoirs for its control.

Another variation would be to employ different capacity reservoirs atdifferent positions (e.g., instead of the herein preferred embodiment,one might have a 2-liter reservoir followed by a one-liter reservoirfollowed by a 3-liter reservoir followed by a one-liter reservoir, andso on).

In principle, the use of individual reservoirs could be abandoned infavor of one multicompartment reservoir consisting of perhaps four totwenty concentric cylinders each activated by solenoids or even bymanual levers external to the temperature-controlled area, all stirredby a single central stir table. Abrupt or step changes in concentrationcould still be accommodated if the stepped change is not delivered viathe stirred central area. The relative positions of the reservoirs couldalso change.

Finally, a fluid metering system could be employed rather than agradient former. In this system, a pump would deliver concentratedcryoprotectant or diluent to a mixing reservoir rather than relying ongravity. This pump would be computer operated to adjust for departuresfrom the programmed concentration. The gradient pump, however, would beretained in order to control overall circuit volume.

The arterial concentration sensor could be located proximal to, ratherthan distal to, the origin of the organ loop in the circuit, but shouldnot be located proximal to the filters.

A pressure sensor to sense pressure developing on the circuit pump sideof the filters could be incorporated as a warning device.

More generally, the device could be separated into two devices, thefirst for preparing organs for cryopreservation and the second forpreparing previously cryopreserved organs for transplantation. The firstdevice would omit R3 and R4 (and associated solenoids) while the secondwould omit R1 and R2 (and associated solenoids) while otherwise beingsubstantially the same as the unified device. Given thatcryopreservation and the recovery from cryopreservation may occur atdifferent locations and under the direction of different individuals,this variation is likely to be of use under practical conditions.Essentially, these two devices would be identical except for the use ofdifferent software and the use of different reservoirs for adding andfor removing cryoprotectant. Another usage could involve the unorthodoxuse of only two reservoirs to accomplish both loading and unloading; forexample, loading could be done using R1 and R3 if only the innercompartment of R3 were used (R3 standing in for R2), and unloading couldbe done using R1 and R3 if R1 substituted for R4.

II. Description of the Methods

A. Preparing an Organ For Cryopreservation and SubsequentTransplantation Into an Animal

The complete cryopreservation method using the above-described apparatuscomprises several parts. One part consists of the pretreatment of thedonor animal and/or the organ prior to its removal from the animal toprepare the organ for its cryopreservation. Another part consists of thechoice of the cryoprotective agents. Another part is the actual protocolfor perfusing the cryoprotectant into the organ prior to itscryopreservation. Another part is the cryopreservation, storage andwarming of the organ using appropriate techniques none of which are partof this invention. Another part of this invention is the protocol forremoving the cryoprotectant(s) from the organ after its warming inpreparation for transplantation into a recipient. Another part istreatment of the organ and the recipient upon organ transplantation.

1. Preteatment of the Donor and the Donated Organ in vivo

The donor, in addition to other standard treatments, received aninfusion of iloprost (Berlex Laboratories, Inc., Cedar Knolls, N.J.)which is a relatively long-lived analog of prostacyclin (PGI₂), or asimilar agent, starting 10 to 20 min. before organ procurement.Applicants have found that iloprost was effective in reducing theapparent toxicity of subsequently-administered cryoprotectant aftereither its intravenous infusion to the systemic circulation or itsadministration directly into the renal artery. The best mode dose ofiloprost was about 25 μg/kg given by either route, although directintra-arterial infusion is presently preferred to maximize organexposure to the agent while minimizing iloprost-mediated systemichypotension. Fifteen μg/kg was also effective, but was less effectivethan 25 μg/kg. Acceptable limits of iloprost concentration for thisapplication are 5-75 μg/kg, depending on species, organ, infusion rate,duration of infusion, etc. Iloprost was typically infused over thecourse of 20 min; acceptable infusion duration limits are 1-60 min forcadaveric organ donors. When hypotension is a limiting factor, iloprostmay be infused at relatively low concentration over a relatively longtime (20-60 min). While not wishing to be bound by any particulartheory, iloprost's protective action may not be a direct cyroprotectiveeffect. The ineffectiveness of iloprost in protecting kidney slices fromcryoprotectant-induced injury suggests that iloprost may simply act as apowerful vasodilator that facilitates uniform cryoprotectantdistribution. Therefore, other vasodilators such as acetylcholine,nitroprusside, nitric oxide, hypertonic and/or hyperoncotic flushsolutions etc., may be substituted for it at doses which producesufficient vasodilation in the organ of interest.

An important option for optimizing results was organ pretreatment withtransforming growth factor beta 1 (TGFβ1), which prevented detachment ofcultured endothelial cells from their substratum in vitro duringsuperfusion with 52% w/v cryoprotectant, when added to the culturemedium at a concentration of 10 ng/ml about 24 hours prior tosuperfusion. The best mode use is to administer a bolus injection ofTGFβ1 of 0.1 μg to 10 μg per kg, 2 to 4 hours before organ donation withor without additional injections at earlier times. The inventors foundthat giving 0.5 μg/kg of human TGFβ1 at 3, 16, and 20 hours before organdonation protected rabbit kidneys from a 40-50 min exposure to 8Mcryoprotectant, thus preventing the otherwise-expected hemorrhage thatresults from such exposure and allowing one animal (exposed for 50 min)to survive until sacrificed on day 15 postoperatively.

After pre-treatment in vivo, the organ of interest was flushed in situwith cold Euro Collins solution, modified UW solution or a comparablyeffective solution in such a manner as to avoid conflicts in multipleorgan procurement. The compositions of these solutions are contained inTable 3. (Should normothermic preservation techniques supersedehypothermic preservation for hearts, the heart can be flushed with warmrather than cold solution.) The flushing solution(s) should initiallycontain iloprost (1 μg/ml in the best mode, acceptable iloprostconcentration limits being 0-10 μg/ml), anticoagulants (e.g., heparin,10,000 units/liter in the present embodiment, acceptable heparinconcentration variations being 500-20,000 units/liter), vasodilators(e.g., papaverine, 40-90 mg/liter in the best mode, 0-90 mg/liter asacceptable limits) and other desired agents. A second flushing solutionshould be used to wash out all of these agents as cooling and bloodwashout is completed. The excised organ (except for organs such as theheart that may be best maintained by normothermic perfusion) should betransferred to an iced bath of flush solution and transported to aperfusion machine capable of introducing and removing cryoprotectants inthe fashion to be described.

                  TABLE 3                                                         ______________________________________                                        Compositions of Perfusion Solutions                                           ______________________________________                                        Compound         mM     g/l                                                   ______________________________________                                        Euro-Collins*                                                                 Dextrose         194    34.96                                                 KH.sub.2 PO.sub.4                                                                              15     2.06                                                  K.sub.2 HPO.sub.4                                                                              42     7.40                                                  KCl              15     1.12                                                  NaHCO.sub.3      10     0.84                                                  RPS-2                                                                         Dextrose         180    32.43                                                 K.sub.2 HPO.sub.4                                                                              7.2    1.25                                                  KCl              28.2   2.11                                                  NaHCO.sub.3      10     0.84                                                  Glutathione      5      1.53                                                  Adenine HCl      1      0.17                                                  CaCl.sub.2       1      0.111                                                 MgCl.sub.2       2      0.407                                                 ______________________________________                                        Modified UW Solution #1                                                                        Modified UW Solution #2                                      Compound mM      g/l     Compound mM    g/l                                   ______________________________________                                        NaH.sub.2 PO.sub.4.H.sub.2 O                                                           25      3.45    NaH.sub.2 PO.sub.4.H.sub.2 O                                                           25    3.45                                  K gluconate                                                                            100     23.42   K gluconate                                                                            100   23.42                                 Mg gluconate                                                                           1       0.21    Mg gluconate                                                                           1     0.21                                  glucose  5       0.90    glucose  15    2.70                                  glutathione                                                                            3       0.92    glutathione                                                                            3     0.92                                  adenosine                                                                              5       1.34    adenosine                                                                              5     1.34                                  HEPES    10      2.38    HEPES    10    2.38                                  adenine  1       0.17    adenine  1     0.17                                  (hydrochloride)          (hydrochloride)                                      ribose   1       0.15    ribose   1     0.15                                  CaCl.sub.2                                                                             0.05    0.0056  CaCl.sub.2                                                                             0.05  0.0056                                HES(g)   --      50      --       --    --                                    ______________________________________                                         *pH = 7.4                                                                     **milliosmolality = 350-365 milliosmolal                                      (Note: RPS2.sup.=  solution is RPS2 without CaCl.sub.2, and also without      MgCl.sub.2)                                                                   (Note: Modified UW Solution #2 does not contain HES but contains more         glucose than modified UW Solution #1)                                    

2. Cryoprotective Agents: Formulae of the Vitrification Solutions V49,V52, V55, V49B and V55B

All perfusion experiments were carried out using solutions designatedhere by V49, V52 and V55 (V49 has sometimes been referred to as VS4. V55has been referred to as VS41A.). At low cooling rates (5°-10° C./min)V49 was found to vitrify at 1,000 atm of applied hydrostatic pressurebut not at ordinary ambient pressures. V52 was inferred to vitrify at500 atmospheres (atm) of applied pressure. V55 was found to vitrify at 1atm.

V49 was composed of dimethyl sulfoxide (D), formamide (F), and1,2-propanediol (P) such that the mole ratio of D to F was 1:1, thetotal mass of D+F+P per liter was 490 grams, and the total mass of P perliter was 150 grams. Thus, per liter, D+F=340 grams, F=124.33 grams, andD=215.67 grams. This mixture of cryoprotectants was preferred based onthe results described below. Acceptable variations for the proportionsof D, F, and P are: D:F weight ratio can be as low as 1.4 and as high as3.5; for the former, the proportion of P:(D+F) should be elevated to18:34 and/or the total concentration raised to 50-51% w/v(grams/deciliter) by the addition of extra P.

The formula for V52 was obtained by multiplying the cryoprotectantcontent of V49 by 52/49, keeping the vehicle solution the same as forV49. The formula for V55 was obtained by multiplying the cryoprotectantcontent of V49 by 55/49, keeping the vehicle solution the same as forV49. Thus, the total concetnration of solutes in V55 was 550 grams/litervs. the 490 grams/liter of V49. V49B was a variation of V49 in which the1,2-propanediol content was replaced gram for gram by 2,3-butanediol(levorotatory form or racemic mixture with less than 5% w/w meso formpresent), and V55B was, similarly, a variation of V55 in which2,3-butanediol replaced the 1,2-propanediol gram for gram. The totalcryoprotectant molarities of V49, V52 and V55 were 7.49, 7.95 and 8.41M,respectively. The molarities of V49B and V55B were slightly lower thanthose of V49 and V55 due to the greater molecular weight of butanediolvs. propanediol.

While not wishing to be bound by any theory, V49 and V55 appear to beparticularly beneficial due to the exceptional ability of formamide topenetrate kidney tissue, the ability of dimethyl sulfoxide to block thetoxicity of formamide, the beneficial balance between the threeingredients (maximizing vitrification tendency while minimizing bothtoxicity and total solute concentration), the lack of a colloid (typicalcolloid concentrations of about 4-7% w/v elevate viscosity), theextraordinarily slow rate of devitrification of these solutions atappropriate pressures (1,000 atm and 1 atm, respectively), and the goodstability of V55 at -135° C. during at least 6 months of storage.

The cryoprotectants used for organ perfusion were adjusted between thelimits represented by V49 and V55, depending upon the pressure to whichthe organ was to be subjected. Balancing an organ's tolerance to highpressures and its tolerance to high cryoprotectant concentrationsallowed optimization of the tradeoff between pressure and concentrationrequired to maintain vitrifiability. For example, an organ that cannottolerate 1,000 atm but that can tolerate 500 atm may be perfused withV52. Concentrations in excess of 550 grams/liter, to a maximum of about600 grams/liter, may be used when heterogeneous nucleation on cooling isa significant problem, since the nucleation process and the growth ofany nucleated ice crystals will be suppressed at these higherconcentrations. One example of a situation in which this problem willarise is the vitrification of very large organs such as the human liverthat will cool particularly slowly. At elevated pressures, similarproportional increases in solute concentration will be required as thecooling rate is lowered.

Experiments (see results below) with kidney slices indicated that V49Bprovided viability identical to the viability obtained with V49. V49Bmay have greater stability than V49. Variations between V49B and V55Bare to be used as per the descriptions above for V49 and V55.

All cryoprotectant solutions must contain, in addition to thecryoprotectants themselves, slowly-penetrating solutes comprising the"carrier" or "vehicle" solution for the cryoprotectants. Typicalexamples would be modified UW solutions, Euro Collins solution, or RenalPreservation Solution 2 (RPS-2) (see Table 3). The best mode method usedEuro Collins as the vehicle solution of choice for kidneys, modified UWsolution (as per Table 3) as the vehicle solution of choice for theliver, and commercial UW solution (Viaspan®) (E. I. DuPont and Nemours)as the vehicle solution of choice for hearts.

3. Protocol for Perfusing the Organ with Cryoprotectant

Typical protocols for cryoprotectant introduction and removal that wereshown to yield reliable, high-quality survival of rabbit kidneys aftercryoprotectant washout, transplantation, and long-term functional andhistological follow-up, are shown in FIGS. 5 and 6 and are additionallydescribed in the flow charts of FIGS. 7A-E. As designated in FIG. 5, theprotocols were divided into at least 7 discrete phases. Phase 1 was anequilibration period during which the organ established stable baselinecharacteristics prior to the introduction of cryoprotectant. Phase 2 wasa gradual increase in cryoprotectant concentration that ended in aconcentration plateau known as phase 3. After spending a certain amountof time in phase 3, during which time the A-V cryoprotectantconcentration gradient usually became approximately zero, theconcentration was stepped to a new plateau, this new plateau phase beingphase 4. As described in more detail below, phase 4 need not be thehighest concentration attained. In FIG. 6, for example, the phase 4concentration is 6.7M, but the final concentration in the experiment ofFIG. 6 was actually 8.4M. Whatever the final concentration, the firstwashout step is indicated in FIG. 5 as phase 5, another concentrationplateau. Phase 6 is the cryoprotectant washout phase and phase 7 is apost-cryoprotectant equilibration phase.

a) Perfusion pressure: The organ was perfused at pressures sufficient toovercome the organ's critical closing pressure but otherwise low enoughto avoid needless damage to the vascular tree. For example, the bestmode perfusion pressure for the rabbit kidney was 40 mm Hg withoutsignificant pulsation. A desirable range of acceptable pressures hasbeen found to be 20-70 mm Hg for different organs and species, includingman, except for the liver. The liver normally receives most of its flowthrough a vein at a pressure typically below 10 mm Hg. Rat liversperfused at 5 mm Hg were able to achieve approximate osmoticequilibration after perfusion with V49 for 20 min when no colloid waspresent, and half of these livers supported life after transplantation.Consequently, the pressure limits for livers are 5-40 mm Hg through theportal vein, and 5-70 mm Hg through the hepatic artery.

b) Initial perfusion (phase 1): In the best mode protocol, perfusion wasfirst carried out for 15 min to establish baseline values for vascularresistance and calibrations (for pressure and refractive index); toensure complete blood washout; and to thermally equilibrate the organ,here the rabbit kidney or the rat liver. Clinically, the initialperfusion time is arbitrary, and can be adjusted (from zero minutes to1-2 days or more) to meet the requirements of the organ procurement andtransportation process. In Applicants' laboratory, the perfusate in thisperiod was Euro Collins solution or RPS-2 for kidneys and a modified UWsolution for livers. However, this initial perfusate could also beanother stabilizing solution in a clinical setting depending upon theneeds of the hospital or procurement team.

c) Initial temperature: The initial perfusion temperature required forthe procurement and transportation of an organ, such as, for example,the kidney, need not be identical to the perfusion temperatureestablished during phase 1. For example, while most organs may beshipped while surrounded by crushed ice at 0 C., other organs may beshipped while being perfused at normothermia (37° C.). When organs areready for cryoprotectant administration, however, a preselected,standardized perfusion temperature is established. In the best mode, theinitial perfusion temperature was 3.5°-4° C., and the acceptable limitswere 0°-15° C. The inventors consider that organs requiring normothermicperfusion for best long-term maintenance can nevertheless be cooled towithin this same temperature range and can be treated in a mannersimilar to that of hypothermically-preserved organs without damagewithin the relatively short times required for this method.

d) Phase 2: Following the initial baseline perfusion, cryoprotectantconcentration was elevated at a constant rate until a first plateau ofconcentration was established. When using a V49-type mixture ofcryoprotectants, the proportions of different cryoprotectants in themixture were held constant while the total concentration was allowed tochange. The rate of increase in total concentration for V49-typesolutions was set to about 51 mM/min (nominally 3M/hr) in the best modefor the kidney, acceptable variations being 31-150 mM/min. These rateswere considerably in excess of the 30 mM/min rates used by knowntechniques for glycerol and propylene glycol which were considered to beunnecessarily and undesirably slow for most applications of the method.Linear elevation of concentration promoted equilibration withoutcreating unnecessarily large osmotic stresses.

e) Temperature reduction during phase 2: The temperature was loweredduring phase 2 to protect the kidney from the chemical toxicity of thecryoprotectant. In the best mode, the temperature reduction began as thearterial cryoprotectant concentration reached 1.3M; acceptable limitsare 0.5M to 3.5M. Temperature descent was terminated as phase 3 wasreached. The concentration change during cooling was about 2.5M in thebest mode but may vary from about 1M to 4.4M.

As noted above, the initial perfusion temperature should be between 0°C. and 15° C. The temperature after cooling should fall within the rangeof -13° C. to +5° C. and the total temperature drop during coolingshould be between 2° C. and 25° C. Cooling should not continue to belowthe freezing point of the organ. In the best mode, the final arterialtemperature was -3° C., representing a fall of 6.5° C. from the initialtemperature and a cooling rate of about 0.33° C./min. The overallcooling rate should not exceed 3° C./min in order to provide adequateopportunity for cryoprotectant diffusion and in order to avoid possiblethermal shock to the organ.

f) Phase 3: The phase 3 plateau was set in the best mode for the kidneyat 25% w/v total cryoprotectant (250 grams/liter, or about 3.8M) when40-49% w/v cryoprotectant was to follow, or 30% w/v, (4.6M) when higherconcentrations (e.g., V55) were to follow, acceptable variations being20-40% w/v or w/w. The phase 3 plateau was set to a level that was closeto half of the phase 4 concentration. Lower phase 3 levels will increaseosmotic stress upon moving to phase 4, whereas substantially higherphase 3 levels will produce increased toxicity due to longer exposuretimes to concentrated cryoprotectant. The duration of phase 3 was set toabout 10 min in the best mode procedure, acceptable variations being5-30 min, depending on perfusion pressure (and thus organ flow rate),vascular resistance, organ permeability to cryoprotectant, and therapidity of toxic responses. The duration was long enough to allow theorgan to at least approximately osmotically equilibrate with thearterial perfusate, as indicated by an arteriovenous concentrationdifference no greater than 50-200M, so as to minimize unnecessaryosmotic stress during the subsequent jump to higher concentrations.

g) Perfusion with vitrification solution by a one-step, two-step orthree-step method: A step change in concentration from phase 3 to phase4 was necessary to control the exposure time to highly concentratedcryoprotectant. The phase 4 concentration may be sufficient forvitrification (a one-step introduction method) or it may be insufficientfor vitrification (requiring one or two additional steps to achievevitrifiability).

The concept behind the two- (and the three-) step approach isillustrated schematically in FIG. 8. In the "one-step" approach, all ofthe cryoprotectant was added in one continuous process (C1), and coolingto cryogenic temperatures then occurred in one step (T1) as well. In the"two-step" approach, part of the cryoprotectant was added in the firststep (C1), and the rest of the cryoprotectant was added in a second step(C2) carried out at temperatures near the freezing point of the solutionused in the first step. In this approach, cooling also took place in twosteps, the first step (T1) having been used to prepare for the secondconcentration increment (C2), and the second step (T2) being used tocool the organ to cryogenic temperatures. In practice, the first coolingstep was preferably to temperatures somewhat above the nominal freezingpoints to guarantee the avoidance of crystallization prior tointroducing higher concentrations of cryoprotectant.

In the best mode, the phase 4 concentration was set to 40% (6.1M) to 44%(6.7M) w/v V49 solutes, a concentration that was not sufficient forvitrification (FIG. 6). Acceptable variations for sub-vitrifiableconcentrations are 30% w/v to 48% w/v V49 solutes or their equivalent.For the one-step introduction, the phase 4 concentration may range from480-600 grams/liter (about 7.4-9.2M) for V49- or V49B-type solutions(for example, see FIG. 5). For non-V49/V49B type solutions, the methodlimits for phase 4 are 35%-60% w/v cryoprotectant.

Phase 4 concentration was held steady for 20 min in the best mode,acceptable variations being 10-60 min. The concentration should be heldsteady long enough for the organ to closely approach osmotic equilibriumwith the perfusate according to the above-described criterion.

For the two step approach, the organ was removed from the perfusionmachine after the completion of phase 4, and was cooled by being placedinto precooled vitrification solution for 5-30 min (5 min in thepreferred mode for rabbit kidneys, longer for more massive organs) priorto being perfused with the vitrification solution. In the best mode, theorgans were cooled toward and subsequently perfused at a temperature of-22°±2° C. (if previously perfused with 6.1M cryoprotectant) or -25°±2°C. (if previously perfused with 6.7M cryoprotectant). The temperaturechosen at this step will be referred to as the "low temperatureperfusion temperature." More generally, the low temperature perfusiontemperature may range from -5° C. to -35° C.

One embodiment of the apparatus used for perfusing organs at thelow-temperature perfusion temperature (to accomplish step C2 in FIG. 8)is illustrated in FIG. 9. In another embodiment, the cooling andlow-temperature perfusion are carried out inside the primary perfusionmachine without substantial operator intervention.

The inventors have perfused rabbit kidneys with V52 at -22° C. or withV55 at -25° C. at a perfusion pressure fluctuating between 20 and 40 mmHg but usually not exceeding 30 mm Hg, having obtained excellent resultsafter subsequent transplantation. Acceptable method limits for perfusionpressure range from 50% to 150% of the previous pressure in theperfusion apparatus for organs other than the liver, or from 50% to 400%of the previous perfusion pressure in the case of the liver.

The time required for equilibration with vitrifiable concentrations atthe low-temperature perfusion temperature was determined in the case ofthe kidney by collecting "urine" produced during the low-temperatureperfusion and determining its osmolality after suitable dilution. Thekidney was deemed to have been equilibrated when the osmolality of theurine approached the osmolality of the arterial perfusate. For otherorgans, the extent of equilibration is determined as usual by thearteriovenous concentration difference. Acceptable equilibration timeswere determined to range from about 20 to about 60 minutes.

Another embodiment that will apply to organs which cannot tolerateexposure to fully vitrifiable solutions at the low-temperature perfusiontemperature is the three-step introduction method. These organs may besuccessfully cryopreserved by perfusing a less-than-fully-vitrifiableconcentration at the low-temperature perfusion temperature (step two),which concentration, being higher than the concentration used prior tocooling to the low-temperature perfusion temperature, will depress thefreezing point of the organ to substantially (i.e., 3° to 20° C.) belowthe low-temperature perfusion temperature. The organ can then beperfused with fully vitrifiable concentrations near the new organfreezing point temperature (step three), at which temperature the fullyvitrifiable concentrations will be sufficiently non-toxic as to betolerated. This embodiment will apply also to organs that require it foravoiding cooling injury.

h) Rationale for the two-step best mode method:

While not wishing to be bound by any theory, the main rationale for thebest mode two-step method was the avoidance of cooling injury.Introducing cryoprotectant at the low-temperature perfusion temperaturewas hypothesized to reduce cryoprotectant toxicity as well.

The inventors discovered that kidneys perfused at -3° C. with V49survived 100% of the time (14 survivors out of 14 perfusions) but whenthey were cooled to -30° C., warmed and washed out using the besttechniques known at the time, the survival rate fell substantially (seeFIG. 13). Kidneys perfused with V52 at -3° C. using the optimaltechniques of the time survived 75% of the time, but when these kidneyswere cooled to -30° C., the survival rate upon warming and washout was0%. Thus, cooling caused injury at 49% w/v cryoprotectant and causedcomplete loss of viability at 52% w/v cryoprotectant. Since, ideally,organs should be preserved in V55 (to avoid the need for highpressures), this trend was unfavorable. However, a positive implicationwas that cooling injury might become negligible at concentrations lowerthan 49%, so that cooling to temperatures near -30° C. might then beinnocuous. This suggested the possibility of cooling at a relatively lowconcentration so as to avoid cooling injury and then raising theconcentration to a vitrifiable level at the lower temperature. Thisapproach would have the additional advantage of exposing the organ tovitrification solution at a temperature at which its toxicity should bereduced. Thus, by avoiding cooling injury, toxicity might also beavoided.

A secondary point was that a variety of experiments on the phenomenon ofthermal shock in both erythrocytes and kidney slices suggested thatcooling injury below -30° C. might be minimal even in the presence ofV55 if cooling injury above -30° C. were first prevented. Therefore, byfirst cooling to near -30° C. in the presence of a concentration thatdoes not cause cooling injury, it was inferred that even V55 might notcause fatal cooling injury when the organ was loaded with V55 at thelow-temperature perfusion temperature and was subsequently cooled tobelow -30° C.

As noted in the preceding section, the first hypothesis was verified inthat the two-step approach successfully avoided cooling injury and thetoxicity of V55 at -25° C. As noted in the results section, the secondhypothesis was also verified in that fatal injury did not occur uponfurther cooling to below -46° C. was also avoided.

4. Cryopreservation of the Organ

The next step of any practical cryopreservation procedure, such asvitrification, is to cool the organ to cryogenic temperatures usingappropriate protocols, with or without prior pressurization. Thecryopreservation step also includes the storage of the organ. Thepresent invention is not concerned with the actual cryopreservation andstorage of the organ, but only with the preparation of the organ forcryopreservation and the preparation of the previously-cryopreservedorgan for transplantation.

5. Perfusion of the Organ in Preparation for its Transplantation

In preparation for transplanting it, the organ is first warmed up fromthe storage temperature to an appropriate temperature for reperfusion ofthe organ. The warming of the organ after its cryopreservation ispresently not performed in the apparatus of this invention. The organmay then be placed back into the perfusion apparatus of this inventionto resume the type of perfusion protocol shown in FIGS. 5 and 6 at thebeginning of phase 5 (the first cryoprotectant washout plateau).

a) Temperature during phase 5: In the best mode method, the organ waswarmed to approximately -3.0° C. and placed into the perfusion apparatusto begin cryoprotectant washout at this temperature. The inventorsunexpectedly found that this approach was superior when the two-stepbest mode method for introducing cryoprotectant was used and wassuccessful even when the final vitrification solution used was V55.Given that the introduction of vitrifiable concentrations was possibleat temperatures near -25° C., the inventors had expected that it wouldbe advantageous as well to remove part of the cryoprotectant at thistemperature in order to avoid the expected high toxicity of fullyvitrifiable concentrations at temperatures near -3° C. Instead, thedilution of vitrification solution at the low temperature perfusiontemperature was found to be detrimental. Within the method limits, thetemperature during phase 5 can range from -20° C. to +5° C.

b) Cryoprotectant concentration and duration of phase 5: Theconcentration of cryoprotectant during phase 5 in the best mode protocolfor the kidney and liver was 30% w/v (300 grams/liter; 4.6M) to 33% w/vV49 solutes (D, F, and P in the usual proportions), acceptablevariations being 20-40% (w/v or w/w) cryoprotectant (roughly 3 to 6.0M).The concentration at this stage should not be less than 40% (2/5) of theconcentration of the vitrification solution in order to avoid osmoticdamage; in the best mode, the concentration at phase 5 was 3/5 of thehighest concentration perfused.

The criterion for terminating phase 5 and moving on to phase 6 wassomewhat different from that previously employed. It was found thatprolonged periods at phase 5 sometimes led to changes suggestive ofcellular uptake of the LMW OBA that was generally present during thisplateau and that should remain extracellular for maintaining theviability of the organ. It was consequently determined that the durationof phase 5 should be limited to what is required to allow the A-Vconcentration difference to begin to return to zero (in the inventors'experience, to return from an off-scale value to a value near -50 mM),rather than prolonged to the point that the A-V concentration differenceis no longer rapidly changing. Note the shorter phase 5 time in FIG. 6as compared to that in FIG. 5, reflecting the optimization required forsuccess at the higher concentrations used in the protocol reflected inFIG. 6. Note also the abrupt end to the recovery of the A-Vconcentration gradient in FIG. 6 as contrasted with the prolongedequilibration of A-V concentration during phase 5 in FIG. 5. For therabbit kidney, the optimal time was determined to be 9 min. Within themethod limits, durations of 0-30 min are acceptable.

c) OBAs and their use during phase 5: One or more OBAs (defined asabove) were generally present during phase 5.

As previously defined, one way to categorize OBAs, for ease ofdiscussion, is as LMW (M_(r) between 100 and 1000 daltons) OBAs and HMW(M_(r) between 1000 and 500,000 daltons) OBAs. However, there is in factno sharp dividing line between LMW and HMW OBAs, and different M_(r)ranges have uniquely different properties, and hence different practicalapplications. Some of these key properties, which give rise to thebroader principles behind the usages described below, can be summarizedas follows:

    ______________________________________                                                Membrane  Osmotic  Oncotic                                            M.sub.r Range                                                                         Permeability                                                                            Effect   Effect                                                                              Viscosity                                                                             Cost                                 ______________________________________                                        180-342 Highest   Highest  Nil   Lowest  Lowest                               343-1,000                                                                             Low to    High-    Nil   Mod.    Mod.-                                        Mod.      Mod.                   High                                 1,000-  Low to Nil                                                                              Low      Nil-  Mod.-   Mod.                                 50,000                     Low   High                                         >50,0000                                                                              Nil       Lowest   Low-  Highest Mod.                                                            High                                               ______________________________________                                    

The inventors have unexpectedly discovered several new modes of OBAusage. For application at phases 5 and 6, these new modes consist of i)combined LMW and HMW OBAs (for use with the highest-concentrationprotocols), ii) single midrange OBAs (for high andmoderate-concentration protocols), iii) very LMW OBAs (forlower-concentration protocols), and iv) specific OBA protocols for theliver. In this section, these usages and the principles on which theydepend are discussed generally without reference to phase 6.

i) Combined LMW and HMW OBAs. For the kidney and most other organs, thebest mode OBA usage was considered to be sucrose, for example about300-350 mM, or other LMW OBA in combination with hydroxyethyl starch(HES: relative molecular mass (M_(r)) of 20-500 kd (20,000-500,000daltons)), for example 3-8% w/v, or other equivalent HMW OBA. Twospecific experimental examples illustrated below which yielded goodresults after perfusion with the normobaric vitrification solution V55involved the use of 350 mM sucrose in combination with 3% w/v HES ofM_(r) 450 kd. Other preferred LMW OBAs include maltose, raffinose,potassium and sodium fructose 1,6-diphosphate, potassium and sodiumlactobionate, potassium and sodium glycerophosphate, potassium andsodium gluconate, maltotriose, maltopentose, stachyose and mannitol. Thepreferred HMW OBA, HES, is sold by McCaw Corp. of Irvine, Calif. as a200 or 450 kd chain, but is easily hydrolyzed to lower molecular weightforms. Particularly preferred are HES molecular weights in the 1 to 100kd range. Other preferred HMW OBAs include polyvinylpyrrolidone (PVP),potassium raft nose undecaacetate (available from Sigma Chemical Co.,St. Louis, Ill.) and Ficoll (1 to 100 kd).

The presence of a LMW OBA is required to counteract the otherwise fatalosmotic effects of a large stepwise drop in penetrating cryoprotectantconcentration. In protocol variations employing larger drops incryoprotectant concentration (e.g., lesser phase 5 concentrations near20% w/v cryoprotectant), more LMW OBA is required (to an upper limit of750 mM). In variations employing higher phase 5 concentrations (e.g.,40% w/v cryoprotectant), less LMW OBA is required (to a lower limit ofabout 150 mM).

This best mode use of OBAs during the first cryoprotectant washoutplateau (phase 5) applies particularly to protocols employing more than7.5M V49 solutes, i.e., to protocols employing less than 500-1,000atmospheres (atm) of hydrostatic pressure for vitrification. Exclusiveuse of the LMW OBAs mannitol and sucrose were found by the inventors tobe compatible with at best only a 30% kidney survival rate (2 survivorsof 7 so treated) when V52 was used in place of V49, vs. a 100% survivalrate (14/14) when V49 was used. However, adding 3% w/v 450 kd HES duringwashout of the cryoprotectant raised survival to 75% when eithermannitol or sucrose was used as the LMW OBA (6 survivors out of 8kidneys treated) when the one-step vitrification solution additionmethod was used.

The concept of using HMW agents as OBs had not previously beencontemplated, at least in part, because such agents have little osmoticeffect in comparison to lower molecular weight compounds. While notwishing to be bound by any specific theory, the following considerationsled the inventors to use HES as a prototypical HMW OBA.

(a) In the presence of high concentrations of cryoprotectant, theconcentration of HMW material was higher with respect to water than inthe case of traditional, dilute aqueous solutions. Therefore, theosmotic effect of the agent was enhanced.

(b) The oncotic function of a HMW agent could be crucial in protectingthe vascular system from abrupt collapse upon sudden dilution of thecryoprotectant or could otherwise benefit the vascular system.

(c) The HMW agent may reduce abnormal cellular uptake of LMW OBAs bylowering interstitial volume (thus lowering the pool size of LMW OBAavailable to penetrate cells) or by acting as a physical barrier todiffusion of LMW OBA to and/or through the cell membrane.

(d) The HMW OBA, by its oncotic action to dilate or prevent the collapseof the vascular compartment, should facilitate cryoprotectant washoutand thus reduce osmotic stress caused by lags in cryoprotectant washout.

(e) Any abnormal increase in membrane permeability that may cause LMWOBAs to partly penetrate organ cells will not cause HMW OBAs topenetrate, thus the use of HMW OBAs will reduce the net amount ofabnormal penetration per miliosmole of OBA that is used.

The best mode use of HMW OBAs was to use agents that have at least theosmotic or oncotic pressure of 3-6% 450 kd HES. However, lower M_(r)agents than this may be better since a relative molecular mass of 50 to200 kd should create equal or greater oncotic pressure and stillguarantee failure of the agent to penetrate a viable cell.

The combination of HMW and LMW OBAs was preferred because the formeroffset the uptake of the latter and added to the latter's osmoticeffectiveness, while LMW agents provided sufficient osmotic pressure toaccomplish the primary job of preventing cellular water uptake duringcryoprotectant dilution. In addition, the high viscosity of HMW OBAs incryoprotectant solutions supported the use of the less viscous LMWagents as the primary osmolytes to which HMW agents were added asadjuncts.

ii) Midrange OBAs. For the kidney and most other organs, anotherpreferred OBA usage method is the exclusive use of single OBAs in themolecular weight range of 360-10,000 daltons, used at totalconcentrations of 2%-15% w/v. Examples of suitable OBAs in thisapplication include maltose, raffinose, potassium and sodium fructose1,6-diphosphate, potassium and sodium lactobionate, maltotriose,maltopentose, stachyose, potassium raffinose undecaacetate, Ficoll andHES within the specified molecular weight range.

While not wishing to be bound by any theory, single agents in thisweight range often adequately combine the properties of LMW and HMW OBAsinto a single agent. Osmolyte impermeability is the most importantfeature of an OBA and this impermeability may approach apractically-relevant maximum at molecular weights between 360-10,000daltons or, more narrowly, of around 360-2,000 daltons. Solutes in thisweight range are relatively osmotically effective while being alsorelatively low in viscosity and relatively high in solubility. Thismiddle molecular weight range is therefore presumptively the ideal onewhen neither oncotic effectiveness nor cost is critical. Some agents inthis weight range will also be impermeable to both kidneys and livers,thus eliminating at least part of the distinction between these organs.

(iii) Very LMW OBAs as the sole OBAs for "low" concentration methods.When the vitrification method was to involve the use of relatively lowconcentrations of cryoprotectant, e.g., V49, the use of mannitol (M_(r)=180 daltons) as the sole OBA has yielded satisfactory results (seeresults section for pertinent data), and the low viscosities of mannitolsolutions maintained better organ flow than more viscous (higher M_(r))solutions. Consequently, another embodiment of the best mode was the useof very LMW OBAs (OBAs with M_(r) ≦400 daltons) as the sole OBAs whenvitrification methods are used that employ elevated pressures and/orconcentrations less than that of V52.

While not wishing to be bound by any theory, lower cryoprotectantconcentrations were less stressful and maintained membrane permeabilitymore effectively, and for this reason allowed lower M_(r) agents to beeffective. Because mannitol was extremely inexpensive and universallynon-toxic and because the cost of OBAs tends to rise sharply with M_(r)in the range from 180-2,000 or more daltons, mannitol and/or similar LMWOBAs (e.g., sucrose, maltose) will be the agents of choice in these"low" concentration embodiments of the method.

(iv) OBA usage for the liver For the liver, the best mode OBA usage wasthe complete omission of OBAs. Two other preferred uses of OBAs are theuse of HMW OBA alone (for example, 3-5% HES, M_(r) 10,000-450,000daltons, or its equivalents as noted above) and the use of midrange OBAs(M_(r) about 350 to 10,000), particularly when the cryoprotectantwashout rate is high.

(a) Complete Omission of OBAs. Experiments with 4 control liversperfused with neither cryoprotectant nor the normal HES of modified UWsolution indicated that life support function could be obtained in threecases. When the experiment was repeated with the inclusion of V49perfusion, and no LMW osmolyte was used, not only did about 50% of thelivers support life after transplantation, but they did so after almostcomplete equilibration with V49, in contrast to livers perfused with V49in the presence of HES, which equilibrated poorly and had a survivalrate no better than the livers perfused without HES. Therefore, neitherLMW nor HMW osmolytes were mandatory for livers.

While not wishing to be bound by any theory, the acceptability and thedesirability of omission of all OBAs for the liver were thought to bebased on the liver's high permeability to both cryoprotectants andnominal LMW OBAs. The liver is unique in that its parenchymal cells areexceptionally permeable to LMW solutes, including cryoprotectants. Thisallows faster rates of cryoprotectant addition and washout with lessosmotic stress than occurs in other organs. For example, liver sliceswere found to withstand abrupt multimolar changes in cryoprotectantconcentration that would have been lethal to most other types of tissue,including the kidney, and using smaller changes in concentration did notproduce improved survival in liver cells after cryoprotectant exposureand washout. With respect to the intact liver, note in FIG. 10 thereasonably steady flow rates (suggesting no excessive osmotic cellswelling) during washout of V49 from the liver despite the absence ofboth LMW OBAs and HMW OBAs. Finally, since liver cells are somewhatpermeable to sucrose, sucrose will be relatively ineffective as an OBduring cryoprotectant washout, but its leakage into the cells mightactually cause cell swelling upon transplantation.

(v) Exclusive Use of HMW OBA. The above-described experiments revealedone difficulty with the omission of HES, and that is the fact that only3 out of 4 control livers (no cryoprotectant) survived perfusion in theabsence of HES, vs. higher survival when HES was used. HES or itsequivalent may therefore have to be present to adequately supporthepatic viability regardless of the presence or absence ofcryoprotectant. Because HES cannot be present (except at minimalconcentrations) during the loading of vitrifiable cryoprotectantconcentrations due in part to its unfavorable effect on viscosity, oneway to maximize HES for maintaining viability would be to add HES onlywhen the cryoprotectant is being washed out, simply because perfusionwith HES will be more feasible from a physical standpoint (lowerviscosity) when the cryoprotectant concentrations are low compared tothe vitrifiable concentrations, and when these concentrations arefalling rather than rising. In this context, HES would not necessarilybe acting as a true OBA but only as an ordinary osmotic support agent.Nevertheless, the HES would be used in essentially the same mannerprocedurally as it would be used if it were being used as an OBA, sofrom a practical point of view this would be the equivalent of using aHMW OBA as the sole OBA. Furthermore, it must be remembered that theliver consists of more than merely hepatocytes, and an osmolyte such asHES could act as a true OBA for these non-hepatocytes. The decision touse HES or other equivalent HMW OBA during elution of cryoprotectantfrom the liver can be made depending on the ability of the type of liverin question to withstand the absence of HES during control perfusionsand to withstand the absence of HES during cryoprotectant elution.

Although the liver did not equilibrate well with cryoprotectant whenperfused with a combination of cryoprotectant and HES in theabove-described experiment, this problem can be overcome by using anosmolyte with a sufficiently low M_(r) to control viscosity adequately,e.g., HMW OBAs equivalent to HES of M_(r) =2-50 kd.

(c) Midrange OBAs for rapid cryoprotectant efflux from the liver. OBAsranging in M_(r) from 350 to 10,000 daltons, being less permeable thansucrose, yet considerably less viscous than most HMW OBAs (hence,perfusable at a sufficiently rapid rate), may protect liver cells otherthan hepatocytes from osmotic injury, especially during very rapid ratesof change of cryoprotectant concentration. Therefore, either one suchagent or a combination of two or more such agents falls within themethod limits for the liver.

d) Phase 6: Gradual reduction of cryoprotectant concentration to zerowith simultaneous elevation of perfusion temperature: In the best modemethod for the kidney, the gradual reduction of cryoprotectantconcentration to zero or virtually zero was carried out at a constantrate of about -42 mM/min (acceptable variations being -31 to -75 mM/minfor the kidney and most other organs, or -31 to -150 mM/min for theliver). Non-constant declining concentration schedules (rapid fall athigh concentrations, slower fall at lower concentrations) are also anacceptable variation, e.g., a linear fall at 1.5 times the averagelinear rate for the first third of the washout followed by a linear fallat 0.86 times the average linear rate for the second two-thirds of thewashout.

During cryoprotectant washout, the temperature was elevated tofacilitate washout, reduce osmotic forces, and restore a perfusiontemperature appropriate for an organ containing no cryoprotectant. Inthe best mode method for the kidney, temperature elevation began as theconcentration fell to 4.7M and continued linearly with concentrationdrop until the initial perfusion temperature was reached and arterialconcentration reached 1.3 to 0.8M (1° C. rise per 0.68 to 0.78M decreasein concentration; total of 3.4-3.9M concentration change during warming)as illustrated in FIGS. 5 and 6. Acceptable variations for theconcentration at which the temperature initially rises are 2.5-5.5M andfor the concentration at which temperature rise is completed are0.5M-4.5M.

e) OB washout during phase 6: The general method for OB washout duringphase 6 was to incompletely wash out the LMW OBA while maintaining HMWOBA concentration (when HMW OBA was present) constant or reducing HMWOBA concentration by only 1-2% w/v. More particularly, as penetratingcryoprotective agent concentrations fell, the concentration of LMW OBAalso fell in proportion reaching a final nonzero concentration of OBwhen penetrating cryoprotectant concentration reached zero. This finalnonzero concentration of LMW OBA was 50 mM in the best mode method andmay acceptably vary from 25 mM to 500 mM. As an example, in the bestmode (FIG. 6), in which 350 mM sucrose was brought to 50 mM sucrosewhile 5.0M cryoprotectant was reduced to 0.0M cryoprotectant at a rateof 42 mM/min, sucrose concentration dropped at the rate of 2.5 mM/min.

While not wishing to be bound by any theory, during reduction ofcryoprotectant concentration, absolute transmembrane osmotic forcesattributable to the cryoprotectant transmembrane concentration gradientbecame reduced, thus reducing the requirement for osmotic buffering.Reducing OB concentration during cryoprotectant washout was thereforedesigned to minimize osmotic damage from the OB both duringcryoprotectant washout and thereafter and was further designed to reducepotential cellular uptake of nominally non-penetrating OBA. No previousperfusion technique of cryoprotectant washout has ever made use of this"declining OB principle." When LMW and HMW OBAs were used together, adifferential decrease in OB was performed wherein the concentration ofthe LMW agents declined while that of the HMW OBAs remained the same ornearly the same.

f) OB washout phase 7:

i) Standard mode. The final step in the method after removing allcryoprotectant is to continue to perfuse the organ to allow it to fullyequilibrate with the cryoprotectant-free medium and, if desired, tocontinue or complete the washout of the OB. Ill the current best modefor the kidney, 50 mM sucrose and 3% w/v HES M_(r) 450 kd was attainedat the end of cryoprotectant washout, and no additional washout of theseOBAs was undertaken prior to transplantation. Although it is acceptableto leave such low concentrations of OB in the organ during short holdingtimes before transplantation, interstitial OB is expected to causeosmotic expansion of the interstitial space during blood reflow with aconsequent temporary reduction in organ perfusion in vivo. This effectwill become unacceptable at higher OB concentrations (≧100-500 mM, or≧3-7% w/v) and will necessitate at least partial OB washout beforetransplantation. A further problem with leaving OB in the organ forextended times before transplantation is the potential leakage of OBinto organ cells with consequent cellular swelling and reduced perfusionupon transplantation. In experiments with V49, the inventors typicallywashed out 50 mM mannitol over the course of 30 min with completesuccess upon transplantation. However, it was generally observed thatleaving 50 mM LMW OB in the kidney for short times beforetransplantation was beneficial at higher cryoprotectant concentrations,in some cases representing the difference between organ survival ordeath. It has never been observed that leaving 50 mM mannitol or sucrosein the kidney prior to transplantation was more detrimental thanentirely removing this final concentration, so the washout of OBA duringphase 7 is primarily concerned with reducing LMW OBA concentrations downto less than about 100-500 mM and with reducing HMW OBA concentrationsdown to less than about 5-8% w/v.

While not wishing to be bound by any theory, the retention of 50 mM LMWOBA is belived to be beneficial because interstitial osmolyte willreduce cell and organelle swelling until the moment metabolism isrestored in vivo, and that metabolizing cells are capable ofosmoregulation to cope with intracellular leaked mannitol or sucroseprovided the extracellular osmolyte can slow down passive cellularswelling long enough for osmoregulation to be restored. In addition, theuse of higher M_(r) OBAs will preclude cellular uptake of OBAs, furtherincreasing the acceptability of leaving in the OBA.

Higher concentrations of OB (up to 500 mM) may be washed out over moreextended times (30-90 min) that depend on the perfusion resistanceresponse to OB dilution. For clinical purposes, the duration of thepost-washout perfusion period, comprising the OB washout, and the degreeof OBA washout must be adjusted to be compatible with the exposure timesimposed by the logistic requirements of organ transportation andtransplantation.

ii) The three-osmolyte washout technique. In the inventors' earlyexperience involving perfusion of 8.4M cryoprotectant at about -3° C.(one-step addition technique), consistent control of vascular resistanceduring cryoprotectant washout and excellent appearance of the kidneys 40minutes after transplantation were obtained when the following procedurewas used, and only when it was used.

After perfusion with 8.4M cryoprotectant, the concentration of thecryoprotectant was dropped to about 5.0M with the simultaneousintroduction of 250 mM sucrose and 4% w/v HES. After a 9 minute phase 5plateau, the standard linear sucrose washout technique was followedwhile holding HES concentration constant at 4% w/v. However, once allcryoprotectant was removed, the HES concentration was gradually reducedto 3% w/v while the sucrose concentration was gradually reduced to zeroand mannitol was concomitantly introduced to a final concentration of 50mM.

Thus, the innovations involved in the three-osmolyte washout techniquewere: 1) to combine a HMW with two LMW OBs resulting in a 3-OBA method,and (2) to replace one OB (sucrose) with another OB (mannitol) justprior to transplantation.

While not wishing to be bound by any theory, this approach was developedfor the following reasons. Sucrose is more effective osmotically thanmannitol, i.e., it is less likely to leak into renal cells due to itshigher molecular weight. However, unlike mannitol, it does not have anyability to quench free radical reactions during reperfusion of the organwith blood upon transplantation. By using sucrose to carry out theprimary osmotic buffering function and mannitol to maintain osmoticbuffering at the end of the perfusion, the advantages of both agentswere obtained and the disadvantages of both agents were avoided. Second,4% HES appeared optimal for balancing the tradeoff between minimizingviscosity while maximizing osmotic and oncotic effectiveness duringphases 5 and 6. Finally, 4% HES was reduced to 3% just beforetransplantation to minimize perfusate viscosity and the quantity ofinterstitial HMW species.

It is not to be construed that this method depends specifically onsucrose, HES or mannitol. The governing principle involved is a generalone.

6. Treatment of the Organ and the Recipient at the Time ofTransplantation and Thereafter

It is important that the recipient receive aspirin (acetylsalicylate,1-3 mg/kg) and heparin (100-250 units/kg) shortly before release of thevascular clamps and reperfusion of the transplanted organ, both higherand lower concentrations of both drugs resulting in vascular obstructionand failure. The best mode concentrations were 2 mg/kg and 200 units/kg,respectively. It may also be helpful to gradually infuse agents thatreverse sulfhydryl oxidation (e.g., aurothioglucose or N-acetylcysteineat serum levels of 0.1-10 mM), inhibit extracellular (e.g., α-2macroglobulin, amiloride, tissue inhibitor of metalloproteinases (TIMP))and intracellular (leupeptin, glycine) proteases or facilitateendothelial cell adhesion (TGFβ1, 0.1-10 μg i.v. per every 5 min for40-300 min). The inventors have found that dimethyl sulfoxide reducesthe ability of renal tissue to restore depleted tissue SH content andhave found massive elevation of urinary urokinase after thetransplantation of rabbit kidneys.

III. Method for the Perfusion of an Organ With Non-CryoprotectantPerfusates

In addition to the organ cryoprotection perfusion protocols, theapparatus and methods described herein are capable of use in a widevariety of protocols for conventional organ hypothermic and normothermicpreservation. In addition, a wide variety of normothermicpharmacological, physiological, and pathophysiological protocols arepossible using the apparatus and methods of this invention. Theinventors indicated many of these possibilities earlier and indescribing the steps required to carry out many of these protocols inFIGS. 11A and 11B, which are self-explanatory.

IV. Results

A. Endothelial cell protection with TGFβ1

TGFβ1 allowed endothelial cells to remain properly attached tofibronectin medium or substrate in a culture flask when washed withcryoprotectant solution Table 4. TGFβ1 is expected to have a similareffect on endothelial cells in vivo.

                  TABLE 4                                                         ______________________________________                                        Protection Against Endothelial Cell Detachment by TGFβ1*                                   # of Non-Detached                                                                          p Value vs.                                    Treatment         Cells        Controls                                       ______________________________________                                        37° C. Controls                                                                          5.05 ± 0.31 × 10.sup.6                                                            --                                              2° C. Controls                                                                          4.33 ± 0.38 × 10.sup.6                                                            n.s.                                           V52 (superfused according to whole                                                              1.38 ± 0.10 × 10.sup.6                                                            <.00002                                        kidney protocol: V52 itself = 20 min                                          exposure)                                                                     V52 + TGFβ1 (same as V52 above                                                             4.95 ± 0.21 × 10.sup.6                                                            n.s.                                           but culture pretreated with TGFβ1                                        at 10 ng/ml for 22 hours)                                                     ______________________________________                                         *Detachment was determined by trypsinizing the flasks after each              experiment, washing out the cultured endothelial cells and counting them.     Detached cells removed during the superfusion are not seen in this assay,     causing the cell count to go down.                                       

B. Rabbit Kidneys.

1. Suitability of V49B-type Solutions.

Viability data from rabbit kidney slices after treatment with V49 orV49B are shown in Table 5.

                  TABLE 5                                                         ______________________________________                                        Viability of Rabbit Kidney Slices Treated With V49 or V49B                                 K/Na ratio of tissue*                                            Treatment    (mean +/- SEM)                                                   ______________________________________                                        V49          3.43 +/- 0.07†                                            V49B         3.27 +/- 0.12†                                            ______________________________________                                         †p > 0.05                                                              *The K/Na ratio was measured after washing out the cryoprotectants and        incubating the cortical slices at 25° C. for 90 minutes to permit      active transport of K.sup.+  and Na.sup.+.                               

2. Suitability of V49 and V52 for the Intact Kidney.

FIG. 12 shows post-operative serum creatinine levels of rabbits whichhad received transplanted kidneys that had been previously perfused withV49 in Euro-Collins solution. Prior to procurement, the kidneys weretreated in vivo with zero, 15, or 25 μg/kg of iloprost administered bysystemic intravenous infusion over a 20 minute period. Kidneys in thesethree groups were exposed to V49 (7.5M) at +2°, 0°-2° and -1° to -6° C.,respectively. Initial and final perfusion temperatures were 2° C. in allcases. Rabbit survivals in these three groups were 5/16 (31%), 6/10(60%), and 10/10 (100%), respectively. Only data for rabbits survivingthe first night after surgery are included. Rabbit survivals dependedentirely on the function of the transplanted kidney because acontralateral nephrectomy was performed at the time of transplantation,and no support by dialysis was attempted. Histology in these rabbits waspoor at long-term follow-up without iloprost treatment, marginal withthe lower dose of iloprost, and normal with the higher dose of iloprostand the lowest perfusion temperatures. The results of control (nocryoprotectant) perfusions with Euro Collins are included in FIG. 8 aswell (bottom curve). Although damage in the best V49 group is greaterthan in the controls, all damage appeared to be fully reversible withina short time postoperatively.

Table 6 shows that when an attempt was made to extend the success at7.5M cryoprotectant to 8M cryoprotectant, the result was nearly uniformfailure unless 3% HES was incorporated into the solutions used to washout the 8M concentration. The use of HES allowed the survival of 75% ofrabbit kidneys after transplantation. Leaving the LMW OBA in the rabbitkidney was also beneficial to the kidneys after their transplantation(Table 6).

                  TABLE 6                                                         ______________________________________                                        Recovery of Whole Rabbit Kidneys                                              Perfused with 8M Cryoprotectant                                                                           % Life                                                                        Support                                           Treatment                   Function                                          ______________________________________                                        A.  Standard Protocol with Either Mannitol or Sucrose                                                         0                                                 Washout                                                                   B.  Modified Protocol with 1st Plateau Raised to 30% and                                                      8                                                 3rd Plateau Raised to 33% w/v to Reduce Osmotic Stress                    C.  Same as B, But Lowered Perfusion Temperature from                                                         29                                                -1.5° C. to -3° C.                                          D.  Same as C, but Used 3% HES During Washout of                                                              75                                                Cryoprotectant and left 50 mM Mannitol in the Kidney                          Until Transplantation                                                     E.  Same as D, but Used Sucrose vs. Mannitol                                                                  75                                            F.  Same as D, but Removed All Mannitol Before                                                                0                                                 Transplantation                                                           G.  Same as E, but Removed All Sucrose Before                                                                 33                                                Transplantation                                                           ______________________________________                                    

C. Overcoming Cooling Injury at -46° C. and Toxicity at 8.4MCryoprotectant

When kidneys were treated with either 7.5M or 8M cryoprotectant using 3%HES during washout as in Table 6, they were still unable to withstandcooling to -30° C. (FIG. 13). The 100% survival rate at 49%cryoprotectant fell to just over 50% as a result of cooling, and the 75%survival rate of the 8M group fell to 0%.

Although some of this injury was due to the greater time required toallow cooling and warming to take place, tissue slice evidence indicatedthat cooling per se was actively detrimental. As seen in FIG. 14,exposure of slices to 8M cryoprotective agent at 0° C. was considerablymore damaging than exposing them to 6.1M cryoprotectant at the sametemperature (cf. bars 2 and 4), and cooling these 8M slices to -23° C.caused additional injury (cf. bars 2 and 3). Interestingly, however,cooling 6.1M slices to -23° C. did not cause additional injury (cf. bars4 and 5). Even more interestingly, when slices loaded with 6.1Mcryoprotectant were transferred to a -23° C. solution of 8M, or even8.4M cryoprotectant, there was still no damage associated with cooling,nor was there damage associated with cooling, nor was there damageassociated with exposure to these higher concentrations (cf. bars 4 and5 to bars 6 and 7). In fact, slices exposed to 8.4M at -23° C. accordingto the two-step approach (first cool, then expose to higherconcentrations, bar 7) had more viability than slices simply exposed tothe lowest concentration of 8M at 0° C. without cooling (bar 2,p=0.033). These results showed that, at least in slices, both coolinginjury and cryoprotectant toxicity were preventable by cooling first ina low concentration and introducing higher concentrations only in asecond step at the lower temperature, in this case -23° C.

Re-examination of FIG. 13 suggested that the same phenomenon applied tothe intact kidney. Recovery was higher at the lower concentration ofcryoprotectant, and if one drew a line connecting the 8M cooled pointand the 7.5M cooled point, it extrapolated to 100% survival at someconcentration below 7.5M. Because the linearity of such an extrapolationwas not known, the inventors elected to try an experiment with aconcentration comfortably below 7.5M, i.e., with 6.1M as in the sliceexperiment.

The results of this experiment are indicated in FIG. 15. 100% of thekidneys loaded with 6.1M cryoprotectant, cooled to around -22° C., andwarmed up (protocol indicated in the insert) supported life, givingexcellent mean serum creatinine levels after 14 days (Cr₁₄) andacceptable peak creatinine values (pCr). FIG. 16 shows the results ofloading 6.1M cryoprotectant at -3° C., cooling to -23° C., and thenperfusing the kidney with 8M cryoprotectant until equilibrium wasachieved. As in the previous experiments, the 8M cryoprotectant waswashed out using 3% HES. In stark contrast to the results of theone-step cryoprotectant addition method followed by cooling to -30° C.(FIG. 13), the 8M kidneys of FIG. 16 had an excellent survival rate of7/8, and the kidneys that did survive were not different from the 6.1Mkidneys in terms of their Cr₁₄ and pCr values, in full agreement withthe slice results of FIG. 14. Furthermore, as shown in FIG. 17, whenkidneys were perfused with 8M cryoprotectant at -22° C., they could thenbe cooled another 10° C. to -32° C. (colder than in FIG. 13), with 100%survival upon warming and with Cr₁₄ values identical to those of slicesexposed only to 6.1M cryoprotectant, again in complete agreement withthe predictions of FIG. 14.

The inset of FIG. 18 shows that the injury associated with coolingincreases between -30° and -60° C. but does not increase with furthercooling to near the glass transition temperature. The main portion ofFIG. 18 shows an attempt to more precisely determine where between -30°and -60° C., cooling injury stops increasing. Although the magnitude ofthe drop was somewhat small in this experiment, it appeared that slicescooled to -45° to -50° C. experienced a maximum amount of coolinginjury.

Using the information of FIG. 18 as a guideline two additionalexperiments were done with intact kidneys. After spending approximately9 months optimizing the procedure for introducing and removing V55, thefollowing optimum method was identified. The first step of the two-stepapproach was to perfuse 44% w/v cryoprotectant (6.73M) at -3° C. andthen cool to -25° C. for perfusion with V55 (55% cryoprotectant, 8.4M).The kidneys were then warmed back to -3° C. and were washed out with 3%w/v HES and 350 mM sucrose as described above. This protocol resulted ina survival thus far of 2 out of 3 kidneys so treated. These kidneyslooked excellent after 40 min of blood reflow in vivo and, as shown inFIG. 19, they were able to return serum creatinine levels to near orbelow 2 mg/dl, an excellent result. Furthermore, one kidney perfusedwith V55 at -25° C. by this procedure was cooled to -46° C. prior towarming and washed by the same procedure used in the non-cooled V55kidneys. The result for this kidney, also shown in FIG. 19 (dashedline), was similar: the kidney looked excellent upon transplantationand, at the time of submission of the patent application, was restoringserum creatinine to a value near 2 mg/dl. The kidney showed a peculiarlydelayed recovery, maintaining creatinine at values near 15 for anunprecedented amount of time, but the peak creatinine and the rate ofreturn of serum creatinine back to baseline after this long delay werenot different than what was observed for the other two V55 kidneys.

Taken together, the slice results of FIG. 18 and the intact kidney dataof FIG. 19 indicated that rabbit kidneys can now be cooled to the glasstransition temperature without losing viability. Furthermore, since FIG.19 employed a concentration of cryoprotectant that vitrifies withoutapplied pressure, the implication is that high pressures are no longermandatory for organ vitrification.

D. Pertinence of Animal Data to Human Kidney Cryopreservation

1. First Human Kidney

A 232 gram human kidney was perfused according to the method of thisinvention and was then vitrified. Digital data from the method wascaptured using a BASIC program and was edited and plotted using asigmaPlot 5.0 graphics package (Jandel Scientific, San Rafael, Calif.)to generate the data in FIGS. 20A, 20B and 21.

The data in FIG. 20A show that the method of the invention produced theexpected results in this human kidney. Although the measured molaritywas slightly greater than the target molarity and the first step changein concentration slightly overshot the target, the data follow theprotocol reasonably well.

The data in FIG. 20B from the same human kidney show that resistance(expressed as mm Hg divided by flow) and flow (ml/min/gm of kidney)behaved in a way that was qualitatively similar to their behavior inrabbit kidneys.

The data from the subsequent vitrification of this human kidneydemonstrated that this method performed adequately. The data in FIG. 21provide no indication of freezing of the kidney which would have beenrepresented by a temperature plateau followed by a relatively rapid fallin temperature. After an initial thermal lag above 0° C. whichrepresented the time for the external temperature front to penetratethrough the mass of the kidney to the temperature probe in the middle,the temperature dropped rather smoothly, revealing virtually no evidencefor ice formation.

2. Second Human Kidney

This human kidney was a pediatric kidney from a four month old donor.This kidney was stored for about 79 hours after it was collected butbefore it was perfused with V55 cryoprotectant according to the methodof this invention. The data in FIG. 22 show the perfusion of this kidneywith V55 (ascending portion of the curve), and the removal of V55cryoprotectant from the kidney (descending portion of the curve). Thedotted and solid lines in FIG. 22 show the achieved and target V55concentrations, respectively. The perfusion pressure was set at 35 mmHgin this experiment.

The discrepancy between the measured and target concentrations wasmerely a matter of calibration rather than a true limitation of themethod. The pressure spikes which occurred when a concentration of 8.4Mwas quickly approached or retreated from reflected software that was notspecifically designed to prevent these spikes and has since beencorrected. This was not a limitation of the method. Since this kidneywas unloaded, a cooling curve was not generated. Resistance, flow andtemperature are not shown in FIG. 22.

Kidney Slice Viability Data

Viability data from rabbit (FIG. 23A) and human (FIG. 23B) kidney slicesand normalized data for rabbit (R) and human (H) kidney slices (FIG.23C) show nearly identical responses of the human and rabbit kidneyslices to V49. Although the data showed a slightly lower recovery ofhuman kidney tissue compared to rabbit tissue after cooling to -30° C.,this recovery was within the variability seen with rabbit kidney slices.The human kidney was several days old before the experiment was carriedout, whereas the rabbit kidneys were "fresh". The absolute human K/Naratio was depressed about as much as would be expected for rabbit slicesstored for a similar time.

These data in combination with the perfusion data in this section showedthat human kidneys can be loaded with cryoprotectant according to themethod of this invention and can be essentially vitrified oncooling--e.g., be cooled below the glass transition temperature withminimal or no ice formation in the organ. These data also showed thatthe cryoprotectant can be removed from the human kidneys using themethod of this invention. Lastly, the similarity of the viability dataof the rabbit and human kidney slices combined with the fact that rabbitkidneys actually survived and maintained the lives of rabbits into whichthey had been transplanted, suggest similar results will be obtainedwhen human kidneys are treated using the methods of this invention.

F. Applicability to Other Organs: The Rat Liver Model

Rat livers were perfused using the protocol as shown in FIG. 10. Theperfusion fluid did not contain either HES or LMW OBs. The data in Table7 show total bile production at 5, 10 and 15 minutes aftertransplantation and survival at 7 days after liver transplantation intohost rats. These data demonstrated that rat livers perfused with thesolutions supported the lives of host rats into which they had beentransplanted after their perfusion.

                  TABLE 7                                                         ______________________________________                                        Functional Recovery and Life Support Function of Rat Livers                   Perfused with Vehicle or V49                                                          Liver     Total Bile Production at 5,                                                                   Rat Survival 7                                      Weight    10 and 15 min (μl/g ± SD)                                                               days after                                  Experiment                                                                            (% change)                                                                              after Transplantation                                                                         Transplantation                             ______________________________________                                        Control Pfn*                                                                          -9.9 ± 1.68 ±                                                                             5.19 ±                                                                           9.32 ±                                                                           6/6 (100%)                              w/UW1.sup.†                                                                    2.1       .94     2.33  3.65                                          (HES)                                                                         Control Pfn*                                                                          -8.7 ± 2.03 ±                                                                             4.62 ±                                                                           7.67 ±                                                                           5/6 (83%)                               w/UW2.sup.‡  (no                                                           0.5       .75     1.50  2.48                                          HES)                                                                          V49 Per-                                                                              8.7 ±  0.66 ±                                                                             1.62 ±                                                                           3.14 ±                                                                           2/4 (50%)                               fusion                                                                        w/UW1.sup.†                                                                    0.7       .50     0.82  1.38                                          V49 Per-                                                                              -6.3 ± 1.20 ±                                                                             2.56 ±                                                                           4.43 ±                                                                           2/4 (50%)                               fusion                                                                        w/UW2.sup.‡                                                                3.0       .98     1.92  3.18                                          (no HES)                                                                      ______________________________________                                         *Pfn = Perfusion                                                              .sup.† UW1 = modified UW Solution 1 (see Table 3)                      .sup.‡ UW2 '2 modified UW Solution 2 (see Table 3)            

Taken together, the data from kidneys and livers implied that theherein-disclosed methods for preparing organs for cryopreservation andof preparing organs for transplantation after cryopreservation arebroadly applicable.

While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexample, and not limitation. Thus the breadth and scope of the presentinvention should not be limited by any of the above described exemplaryembodiments, but should be defined only in accordance with the followingclaims and their equivalents. Since it will be understood by those ofskill in the art that various changes in form and detail may be madetherein without departing from the spirit and scope of the invention,this application is intended to cover any variations, uses, oradaptations of the invention following, in general, the principles ofthe invention and including such departures from the present disclosureas come within known or customary practice within the art to which theinvention pertains and as may be applied to the essential featureshereinbefore set forth and as follows in the scope of the appendedclaims.

What is claimed is:
 1. A method of preparing a biological organ forcryopreservation, comprising:(a) flushing the organ with acryoprotectant-free, stabilizing flushing solution; (b) perfusing thecryoprotectant-free organ of step (a) with a perfusion solution undercontrolled conditions in which the concentration of cryoprotectant isgradually increased in the perfusion solution to a first predeterminedconcentration, while the temperature of the organ and the perfusionsolution is concurrently reduced from an initial temperature of about 0°to 37° C. to a lower temperature of about -13° to +5° C.; (c)maintaining the concentration of the cryoprotectant for a sufficienttime to permit the approximate osmotic equilibration of the organ tooccur; (d) increasing the concentration of cryoprotectant in theperfusion solution, from the concentration in step (c), under controlledconditions to a first intermediate concentration, which is notsufficient for vitrification, and maintaining the concentration ofcryoprotectant at that intermediate concentration for sufficient time topermit the approximate osmotic equilibration of the organ; (e) reducingthe temperature of the organ to a temperature below that of step (b);and (f) increasing the concentration of cryoprotectant in the perfusionsolution, to a level sufficient for vitrification.
 2. The method ofclaim 1, further comprising treating the organ in vivo with at least onevasodilator prior to flushing step (a).
 3. The method of claim 2,wherein the organ is further exposed to at least one vasodilator duringflushing step (a).
 4. The method of claim 3, wherein the vasodilator isselected from the group consisting of iloprost, acetylcholine,nitroprusside, nitric oxide, papaverine, osmotic agents, and oncoticagents.
 5. The method of claim 2, wherein the vasodilator is selectedfrom the group consisting of iloprost, acetylcholine, nitroprusside,nitric oxide, papaverine, osmotic agents, and oncotic agents.
 6. Themethod of claim 1, wherein at least one vasodilator is present in theflushing solution of step (a).
 7. The method of claim 6, wherein thevasodilator is selected from the group consisting of iloprost,acetylcholine, nitroprusside, nitric oxide, papaverine, osmotic agents,and oncotic agents.
 8. The method of claim 1, further comprisingtreating the organ in vivo with transforming growth factor β1, prior tostep (a).
 9. The method of claim 1, wherein transforming growth factorβ1 is included in the flush solution of step (a).
 10. The method ofclaim 1, wherein the organ is exposed to transforming growth factor β1both in vivo prior to step (a), and its vitro during flushing step (a).11. The method of claim 1, further comprising exposing the organ to bothtransforming growth factor β1 and at least one vasodilator, wherein theexposure occurs in vivo prior to step (a), or during flushing step (a),or both.
 12. The method of claim 1, wherein the concentration ofcryoprotectant in step (f) is sufficient to permit vitrification in theabsence of applied hydrostatic pressure.
 13. The method of claim 1,wherein the magnitude of the concentration of cryoprotectant in steps(b) and (d), and the temperature of steps (b) and (e), is such thatcooling injury of the organ is avoided or minimized in steps (b) and(e), and such that cryoprotectant toxicity is avoided or minimized insteps (b), (d), and (f).
 14. A method of preparing a biological organfor cryopresentation, comprising:(a) flushing the organ with acryoprotectant-free, stabilizing flushing solution; (b) perfusing thecryoprotectant-free organ of step (a) with a perfusion solution undercontrolled conditions in which the concentration of cryoprotectant isgradually increased in the perfusion solution to a first predeterminedconcentration, while the temperature of the organ and the perfusionsolution is concurrently reduced from an initial temperature of about 0°to 37° C. to a lower temperature of about -13° to +5° C.; (c)maintaining the concentration of the cryoprotectant for a sufficienttime to permit the approximate osmotic equilibration of the organ tooccur; (d) increasing the concentration of cryoprotectant in theperfusion solution, from the concentration in step (c), under controlledconditions to a first intermediate concentration, which is notsufficient for vitrification, and maintaining the concentration ofcryoprotectant at that intermediate concentration for sufficient time topermit the approximate osmotic equilibration of the organ; (e) reducingthe temperature of the organ to a temperature below that of step (b);(f) further increasing the concentration of cryoprotectant in theperfusion solution, from the concentration in step (d), under controlledconditions to a second intermediate concentration, which is notsufficient for vitrification, and maintaining the concentration ofcryoprotectant at that intermediate concentration for sufficient time topermit the approximate osmotic equilibration of the organ; (g) furtherreducing the temperature of the organ to a temperature below that ofstep (e) wherein the temperature is about -5° to about -35° C.; and (h)increasing the concentration of cryoprotectant in the perfusionsolution, to a level sufficient for vitrification.
 15. The method ofclaim 14, further comprising exposing the organ to either transforminggrowth factor β1 or at least one vasodilator, wherein the exposureoccurs in vivo prior to step (a), or during flushing step (a), or both.16. The method of claim 14, further comprising exposing the organ totransforming growth factor β1 and to at least one vasodilator, whereinthe exposure occurs in vivo prior to step (a), or during flushing-step(a), or both.
 17. The method of claim 1 or 14, wherein the organ is akidney, liver or heart.